Method for rapidly extracting mRNA from mRNA-LNP

Abstract

The invention discloses a method for rapidly extracting mRNA from mRNA-LNP, which comprises the following steps: adding a polar organic solvent and a non-polar organic solvent into an mRNA-LNP sample to obtain a mixed solution; standing or centrifuging the mixed solution, and taking an upper-layer solution to obtain an mRNA (messenger Ribonucleic Acid) solution; wherein the polar organic solvent can be selected from at least one of alcohols, ketones and amides; the non-polar organic solvent can be selected from at least one of alkanes, halogenated hydrocarbons and esters. By utilizing the method provided by the invention, the mRNA can be quickly and efficiently extracted from the mRNA-LNP, the operation is simple, and the sample loss is small.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology and relates to a method for rapidly extracting mRNA from mRNA-LNP. Background Technology

[0002] Currently, there are two main methods for extracting mRNA from mRNA-LNP: Method 1: Isopropanol precipitation method for mRNA extraction from mRNA-LNP: Add 100 µL of mRNA-LNP sample to 900 µL of 100% isopropanol containing 60 mM ammonium acetate for a 10-fold dilution. Vortex to mix thoroughly. Incubate the sample at 4°C with a concentration of 14000 × 10⁻⁶. g Centrifuge for 15 minutes. Discard the supernatant, then wash the precipitate with 1 mL of 100% isopropanol. Vortex the sample and centrifuge again at 14000 × 10⁻⁶ at 4°C. g Centrifuge for 15 minutes. Wash the mRNA precipitate with 70% ethanol, then dry the sample at room temperature using a vacuum centrifuge for 20 minutes. The dried sample can be stored or reconstituted with 100 µL of RNase-free water at room temperature for quantitative analysis by UV absorbance.

[0003] Method 2: Chemical reagent mixture precipitation method for mRNA-LNP extraction: Add 10 µL of 1% Triton X-100 to 100 µL of mRNA-LNP sample and incubate with shaking at 50°C for 10 minutes. Next, add 900 µL of a mixture of phenol / chloroform / isoamyl alcohol in a volume ratio of 25:24:1. Then, precipitate the extract by adding 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volume of 100% ethanol. Incubate at -20°C for 12 hours. Then, incubate at 4°C with a concentration of 12000 × 10⁻⁶. g The sample was centrifuged for 10 minutes. The precipitate was washed with 70% ethanol, and then dried in a vacuum centrifuge for 20 minutes at room temperature. The dried sample could be stored or reconstituted with 100 µL of RNase-free water at room temperature for quantitative analysis by UV spectrophotometry.

[0004] The methods described above all use organic reagents to destroy mRNA-LNPs, releasing the mRNA from the LNPs and causing it to precipitate. The mRNA in the mRNA-LNPs is then obtained through centrifugation, washing, and drying. These methods are time-consuming, complex, and result in significant sample loss. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapidly extracting mRNA from mRNA-LNP, thereby solving at least one of the above-mentioned technical problems.

[0006] According to one aspect of the present invention, a method for rapidly extracting mRNA from mRNA-LNP is provided, comprising the following steps: A polar organic solvent and a non-polar organic solvent are added to the mRNA-LNP sample to obtain a mixture; After the mixture has been left to stand or centrifuged, the supernatant is collected to obtain the mRNA solution. The polar organic solvent can be selected from at least one of alcohols, ketones, and amides; Nonpolar organic solvents may be selected from at least one of alkanes, halogenated hydrocarbons, and esters.

[0007] mRNA-LNP samples contain a large amount of lipid components, and most lipid compounds are poorly soluble in water. Phosphate-containing lipid compounds such as DSPC, DOPC, and DSPG have some solubility in both highly polar and non-polar organic solvents, but their solubility in polar organic solvents is stronger than in non-polar ones. Similarly, sterols such as cholesterol also have some solubility in both polar and non-polar organic solvents, but their solubility in non-polar organic solvents is stronger. According to the principle of "like dissolves like," a single solvent cannot meet the solubility requirements of the various lipid components in mRNA-LNP.

[0008] To achieve rapid and efficient extraction of mRNA from mRNA-LNP, this invention involved extensive experimentation. The study ultimately discovered that a specific ratio of polar solvent, non-polar solvent, and water creates a mixture optimal for dissolving various lipid components in mRNA-LNP. When the polar solvent, non-polar solvent, and mRNA-LNP sample are mixed, differences in solubility and density among these three components cause the solution to separate into two layers. The lower layer is primarily composed of non-polar organic solvents (such as chloroform and dichloromethane), but also contains dissolved polar organic solvents (such as methanol, ethanol, and isopropanol) and water from the mRNA-LNP sample. By adjusting the volume ratio of the polar solvent, non-polar solvent, and mRNA-LNP sample, a lower layer highly favorable for lipid dissolution can be obtained, allowing the lipid components in the mRNA-LNP sample to be extracted into this lower layer. The upper layer is primarily water, mixed with small amounts of non-polar and polar organic solvents. The water-soluble components of the mRNA-LNP are naturally extracted into the upper layer. In addition to mRNA, the water-soluble components in mRNA-LNP also include Tri, PBS buffer, and sucrose. These components maintain a favorable pH environment (7-8) and a certain ionic strength in the upper layer solution. In this neutral to slightly alkaline solution environment, the phosphate groups in mRNA are fully dissociated, resulting in strong electrostatic repulsion between molecules and stable hydrogen bonding with water molecules. At the same time, the upper mixed solution has a certain ionic strength, which prevents excessive stretching of mRNA molecules and increased viscosity, thus facilitating the extraction of mRNA into the aqueous layer.

[0009] In some embodiments, the polar organic solvent may be selected from at least one of alcoholic organic solvents.

[0010] In some embodiments, the nonpolar organic solvent may be selected from at least one of halogenated hydrocarbon organic solvents.

[0011] In some embodiments, the polar organic solvent may be selected from at least one of methanol, ethanol, isopropanol, and butanol.

[0012] In some embodiments, the nonpolar organic solvent may be selected from dichloromethane, trichloromethane, or combinations thereof.

[0013] In some implementations, the concentration of total lipids in the mRNA-LNP sample can be 1.8–36 mg / mL.

[0014] In some embodiments, the mass ratio (drug-to-liquid ratio) of mRNA to total lipids in the mRNA-LNP sample is 1:5 to 1:60. Preferably, the drug-to-lipid ratio is 1:10 to 1:40. More preferably, the drug-to-lipid ratio is 1:10, 1:15, 1:20, and 1:40.

[0015] In some implementations, the volume ratio of the mRNA-LNP sample, the polar organic solvent, and the non-polar organic solvent can be 1:(1~1.5):(0.8~6).

[0016] In some embodiments, when the polar organic solvent is methanol and the nonpolar organic solvent is dichloromethane, the volume ratio of the mRNA-LNP sample, the polar organic solvent, and the nonpolar organic solvent can be 1:(1~1.5):(0.8~6). Preferably, when the volume ratio of the mRNA-LNP sample, the polar organic solvent, and the nonpolar organic solvent is in the range of 1:1:2~1:1.5:2, 1:1:1~1:1:3, 1:1.2:0.8~1:1.2:3.5, or 1:1.5:1.5~1:1.5:6, a higher extraction rate of mRNA can be obtained.

[0017] In some embodiments, when the polar organic solvent is ethanol and the nonpolar organic solvent is dichloromethane, the volume ratio of the mRNA-LNP sample, the polar organic solvent, and the nonpolar organic solvent can be 1:(1~1.5):(1~3). Preferably, when the volume ratio of the mRNA-LNP sample, the polar organic solvent, and the nonpolar organic solvent is in the range of 1:1:2~1:1.5:2, 1:1:1~1:1:3, 1:1.2:1~1:1.2:3, or 1:1.5:1~1:1.5:3, a higher extraction rate of mRNA can be obtained.

[0018] In some embodiments, when the polar organic solvent is isopropanol and the nonpolar organic solvent is dichloromethane, the volume ratio of the mRNA-LNP sample, the polar organic solvent, and the nonpolar organic solvent can be 1:(1~1.5):(1~3). Preferably, when the volume ratio of the mRNA-LNP sample, the polar organic solvent, and the nonpolar organic solvent is in the range of 1:1:2~1:1.5:2, 1:1:1~1:1:3, 1:1.2:1~1:1.2:3, or 1:1.5:1.5~1:1.5:3, a higher extraction rate of mRNA can be obtained.

[0019] In some implementations, the lipid component of the mRNA-LNP sample includes neutral lipids.

[0020] In some embodiments, neutral lipids may include phosphate-containing neutral lipids and sterol lipids.

[0021] In some embodiments, neutral lipids containing phosphate groups include, but are not limited to, distearylphosphatidylcholine (DSPC), egg yolk lecithin (EPC), soybean lecithin, hydrogenated soybean lecithin (HSPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoylphosphatidylcholine (DOPC), and arachidoylphosphatidylcholine (DAPC). One or more of the following: dimyristoyl phosphatidylethanolamine (DMPE), dilauryl phosphatidylethanolamine (DLPE), distearatel phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoyl ethanolamine (POPE), 1,2-distearatel-sn-glycerol-3-phosphatidylglycerol (DSPG), and 1-hexadecane-2-(cis-9-octadecenoyl)-sn-glycerol-3-phosphocholine (POPC).

[0022] In some embodiments, sterol lipids include, but are not limited to, one or more of β-sitosterol, stigmasterol, campesterol, cholesterol, and sterol derivatives.

[0023] In some implementations, the steroid lipid is cholesterol.

[0024] In some embodiments, the lipid component of the mRNA-LNP sample includes ionizable lipids, wherein the ionizable lipids include, but are not limited to, one or more of tertiary amines and their derivatives, pyrrolidines and their derivatives, piperazines and their derivatives, and piperidines and their derivatives, such as: DODMA (1,2-dioleyloxy-3-dimethylaminopropane, 1,2-dioleyl-3-dimethylaminopropane, CAS: 104162-47-2), ALC-0159 (m-DTDAM-2000), A066 (Z016) (1-(2,3-bis(((9Z, 12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine), L-319 (CAS: 1351586-50-9), DODAP, C12-200 (CAS: 1220890-25-4), 5A2-SC8 (CAS: 1857341-90-2), 306Oi10 (CAS: 2322290-93-5), 306-O12B-3 (CAS: 2982842-90-8), Moderna Lipid5, AcuitasA9 (CAS: 2036272-50-9), ALC-0315 (CAS: 2036272-55-4), SM-102 (also known as HUO, Moderna Lipid H, 1-octylnonyl) 58-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate), DLin-MC3-DMA((6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate), DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLinDMA, other DLinDMA classes, other DLin-K-DMA classes, lipid 5A2-SC8.

[0025] In some embodiments, the lipid component of the mRNA-LNP sample includes polyethylene glycol lipids; wherein the polyethylene glycol lipids include, but are not limited to, one or more of PEG-docoalkylacetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-phospholipid, PEG-ceramide, DMG-PEG (also known as mPEG-DMG), DSPE-PEG, and DSG-PEG.

[0026] In some embodiments, the lipid component of the mRNA-LNP sample may further include permanently positively charged cationic lipids, including but not limited to quaternary ammonium cationic lipids. When the lipid component of the mRNA-LNP sample contains permanently positively charged cationic lipids, during LNP dissolution, the permanently positively charged cationic lipids, such as DOTAP, will have their hydrophilic N-terminal groups... + It can form strong ion-dipole interactions with the OH (dipole negative end) of polar organic solvents (such as methanol) and the OH (dipole negative end) of water, competitively capturing DOTAP bound to mRNA. This not only promotes the dissolution of DOTAP but also promotes the release of mRNA into the aqueous phase, thereby increasing the extraction rate.

[0027] In some embodiments, permanent cationic lipids include, but are not limited to, DOTMA (CAS: 104872-42-6), DDBA, DMRIE, DOTAP (CAS: 132172-61-3, (2,3-Dioleoyloxy-propyl)-trimethylammonium-chloride), DOTIM, SAINT, DC-Chol (DC cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), BGTC, CTAP, DOSPA, DORIE, DODAB, DOIC, DMEPC, DOGS, DIMRI, DC-6-14, CLIP 1, DORIE, DOSPA, CLIP 6, and CLIP. 9, and one or more of those disclosed in US Patent 5,049,386, International Publications WO91 / 16024, WO97 / 019675, WO2005 / 121348, WO2009 / 086558 and WO2011 / 13636.

[0028] In some embodiments, the lipid components of the mRNA-LNP sample include ionizable lipids, phosphate-containing neutral lipids, cholesterol, and polyethylene glycol lipids, or are composed of the same. The molar ratio (mol%) of ionizable lipids: phosphate-containing neutral lipids: cholesterol: polyethylene glycol lipids is (30~70): (10~40): (20~50): (1~20).

[0029] In some embodiments, the lipid component of the mRNA-LNP sample consists of DODMA, DSPC, cholesterol, and mPEG-DMG-2K in a molar ratio of 50:10:38:2.

[0030] In some embodiments, the lipid component of the mRNA-LNP sample consists of SM-102, DSPC, cholesterol, and mPEG-DMG-2K in a molar ratio of 50:10:38:2.

[0031] In some implementation methods, the lipid components of the mRNA-LNP sample include permanent cationic lipids, ionizable lipids, phosphate-containing neutral lipids, cholesterol, and polyethylene glycol lipids, or a combination thereof. The ratio of permanent cationic lipids to ionizable lipids to phosphate-containing neutral lipids to cholesterol to polyethylene glycol lipids is (1–20): (30–50): (10–40): (10–40): (1–20) based on the total lipid molar ratio (mol%).

[0032] In some embodiments, the lipid component of the mRNA-LNP sample consists of DOTAP, SM-102, DSPC, cholesterol, and mPEG-DMG-2K in a molar ratio of 10:40:10:38:2.

[0033] In some embodiments, the lipid component of the mRNA-LNP sample consists of DOTAP, DODMA, DOPC, cholesterol, and mPEG-DMG-2K in a molar ratio of 5:40:27.5:20:7.5.

[0034] In some embodiments, the lipid component of the mRNA-LNP sample consists of DOTAP, DODMA, DOPC, cholesterol, and mPEG-DMG-2K in a molar ratio of 15:25:20:38.5:1.5.

[0035] In some implementations, the centrifugation speed can be (0~12000) rpm, and the centrifugation time can be (1~10) min. Centrifugation time and centrifugation speed have no significant effect on the extraction effect of mRNA. The mixture can be separated into upper and lower layers by standing for 10 min or centrifuging at 4000~12000 rpm for 1~5 min.

[0036] In some implementations, the centrifugation speed can be 12,000 rpm and the centrifugation time can be 2 minutes. This not only effectively saves time but also achieves a high mRNA extraction rate.

[0037] The beneficial effects of this invention include: (1) The method provided by the present invention can quickly and efficiently extract mRNA from mRNA-LNP, and can complete the extraction of mRNA in as little as 10 minutes.

[0038] (2) The method provided by the present invention only requires simple reagent addition and centrifugation operations, and the operation is simple.

[0039] (3) The method provided by the present invention has a high mRNA recovery rate (about 100%), low sample loss, and the integrity of the extracted mRNA is consistent with that of the original mRNA solution. The extracted mRNA can be used for integrity, fragment percentage and sequence identification analysis, etc., which can better monitor the quality of mRNA-LNP products and assist in the research of mRNA-LNP formulation formulation process.

[0040] (4) The method provided by the present invention is applicable to the extraction of mRNA from mRNA-LNP samples with different lipid compositions. It has a wide range of applications and is especially applicable to mRNA-LNP samples with permanent cationic lipids in the lipid components. Attached Figure Description

[0041] Figure 1 Capillary electrophoresis pattern for detecting the integrity of mRNA stock solution; Figure 2 This is a capillary electrophoresis pattern used in Example 2 to detect the integrity of mRNA extracted with an extractant consisting of 120 μL methanol and 200 μL dichloromethane. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0043] Example 1 The preparation method for mRNA-LNP samples includes the following steps: Weigh SM-102, DOTAP, DSPC, cholesterol, and mPEG-DMG-2K, dissolve them in ethanol to prepare a total lipid solution of 20 mg / mL, which is the ethanol phase; dissolve the mRNA stock solution in sodium acetate buffer to prepare a 0.33 mg / mL mRNA sodium acetate solution (25 mmol / L), which is the aqueous phase; mix the two phases using a liquid phase pump, and then use Tris-sucrose solution for tangential flow ultrafiltration to obtain mRNA-LNP.

[0044] Specifically, the preparation method of mRNA-LNP samples may include the following steps: (1) Preparation of sodium acetate buffer: Weigh an appropriate amount of sodium acetate, dissolve it in water, and adjust the pH to 5.2 with acetic acid to prepare a 50 mmol / L sodium acetate buffer (pH 5.2).

[0045] (2) Preparation of lipid solution: Weigh appropriate amounts of SM-102, DOTAP, DSPC, cholesterol and mPEG-DMG-2K in the molar ratio (40:10:10:38:2), dissolve in ethanol, bathe in a 40℃ water bath until the lipids are completely dissolved, add ethanol to make up to a volume of 20mg / mL lipid solution.

[0046] (3) Preparation of Tris-sucrose solution: Weigh appropriate amounts of Tris and sucrose, dissolve them in water, adjust the pH to 7.5 with hydrochloric acid, and prepare a Tris-sucrose solution of 25 mmol / L Tris - 300 mmol / L sucrose (pH 7.5).

[0047] (4) LNP self-assembly: 1) Add an appropriate amount of the stock solution of mRNA with substitution number ZJHC001 (its nucleotide sequence is shown in SEQ ID NO:24 in patent CN117625652A) to 50 mmol / L sodium acetate buffer, dilute with water to prepare an mRNA sodium acetate solution of about 0.33 mg / mL (25 mmol / L), and preheat in a water bath at 40℃; 2) Take an appropriate amount of lipid solution and preheat it in a water bath at 40℃; 3) Using a liquid phase pump, the lipid solution and the sodium acetate solution of mRNA are rapidly mixed in a T-type flow rate ratio of 1:3 to form an LNP solution through self-assembly; 4) After mixing, add 4 times the volume of Tris-sucrose solution to the LNP solution formed in step 3).

[0048] (5) Ultrafiltration: Using Tris-sucrose solution as the replacement solution, the hollow fiber column was used for tangential flow ultrafiltration 5 times to obtain mRNA-LNP.

[0049] The obtained mRNA-LNP contained approximately 3.6 mg / mL of total lipids and approximately 180 μg / mL of mRNA, with a drug-to-lipid ratio of 1:20.

[0050] Example 2 (1) When the sample size is 100 μL and the amount of dichloromethane is 200 μL, the effect of methanol dosage on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 portions. 200 μL of dichloromethane was added to each portion, followed by different amounts of methanol (80 μL, 100 μL, 120 μL, 150 μL, and 200 μL). Each methanol amount was prepared in triplicate. The mixtures were then centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (Thermo Fisher, NANODROP ONE). The effect of different amounts of methanol and 200 μL of dichloromethane as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 1.

[0051] Table 1. Extraction effect of different amounts of methanol and 200 μL dichloromethane on mRNA.

[0052] As shown in Table 1, when the sample volume is 100 μL and the dichloromethane volume is 200 μL, the methanol volume is in the range of 100 μL to 150 μL, that is, when the sample-methanol-dichloromethane volume ratio is 1:1:2 to 1:1.5:2, good mRNA extraction results can be obtained.

[0053] (2) When the sample size is 100 μL and the methanol volume is 100 μL, the effect of dichloromethane volume on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured, and 24 samples were prepared. 100 μL of methanol was added to each sample, followed by different amounts of dichloromethane (50 μL, 80 μL, 100 μL, 150 μL, 250 μL, 300 μL, 350 μL, and 400 μL). Each amount of dichloromethane was prepared in triplicate. The samples were mixed, centrifuged at 12000 rpm for 2 min, and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (Thermo Fisher, NANODROP ONE) to investigate the effect of different amounts of dichloromethane and 100 μL of methanol as the extraction solvent on the mRNA extraction efficiency. The results are shown in Table 2.

[0054] Table 2. Extraction effect of different amounts of dichloromethane and 100 μL methanol on mRNA.

[0055] As shown in Table 2, when the sample volume is 100 μL and the methanol volume is 100 μL, the dichloromethane volume is in the range of 100 μL to 300 μL, that is, when the volume ratio of sample-methanol-dichloromethane is 1:1:1 to 1:1:3, good mRNA extraction results can be obtained.

[0056] (3) When the sample size is 100 μL and the methanol volume is 120 μL, the effect of dichloromethane volume on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured, and 27 samples were prepared. 120 μL of methanol was added to each sample, followed by different amounts of dichloromethane (50 μL, 80 μL, 100 μL, 150 μL, 250 μL, 300 μL, 350 μL, 400 μL, and 450 μL). Each amount of dichloromethane was prepared in triplicate. The samples were mixed and centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (Thermo Fisher, NANODROP ONE). The effect of different amounts of dichloromethane and 120 μL of methanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 3.

[0057] Table 3. Extraction effect of different amounts of dichloromethane and 120 μL methanol on mRNA.

[0058] As shown in Table 3, when the sample volume is 100 μL and the methanol volume is 120 μL, the dichloromethane volume used is in the range of 80 μL to 350 μL, that is, when the volume ratio of sample-methanol-dichloromethane is 1:1.2:0.8 to 1:1.2:3.5, good mRNA extraction results can be obtained.

[0059] (4) When the sample size is 100 μL and the methanol volume is 150 μL, the effect of dichloromethane volume on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 30 portions. 150 μL of methanol was added to each portion, followed by different amounts of dichloromethane (50 μL, 100 μL, 150 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, and 600 μL). Each amount of dichloromethane was prepared in triplicate. The mixtures were then centrifuged at 12,000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (Thermo Fisher, NANODROP ONE). The effect of different amounts of dichloromethane and 150 μL of methanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 4.

[0060] Table 4. Extraction effect of different amounts of dichloromethane and 150 μL methanol on mRNA.

[0061] As shown in Table 4, when the sample volume is 100 μL and the methanol volume is 150 μL, the dichloromethane volume should be in the range of 150 μL to 600 μL, i.e., the sample-methanol-dichloromethane volume ratio should be 1:1.5:1.5 to 1:1.5:6, to achieve good mRNA extraction results.

[0062] Example 3: Investigating the effects of centrifugation time and speed on mRNA extraction efficiency. Twelve samples of mRNA-LNP prepared in Example 1 were prepared, each containing 100 μL of methanol and 200 μL of dichloromethane. The centrifugation time and speed were set to 10 min (0 min), 12000 rpm for 1 min, 5 min and 8 min respectively. The effects of centrifugation time and speed on mRNA extraction rate were investigated. The results are shown in Table 5.

[0063] Table 5. Effects of centrifugation time and speed on mRNA extraction efficiency

[0064] As shown in Table 5, centrifugation time and centrifugation speed have no effect on the extraction effect of mRNA. When the speed is 0, it needs to be left to stand for a long time. Therefore, centrifugation at 12000 rpm for 2 min is preferred as the rapid extraction condition.

[0065] Example 4 (1) When the sample volume is 100 μL and the dichloromethane volume is 200 μL, the effect of ethanol volume on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 portions. 200 μL of dichloromethane was added to each portion, followed by different amounts of ethanol (80 μL, 100 μL, 120 μL, 150 μL, and 200 μL). Each ethanol volume was prepared in triplicate. The mixtures were then centrifuged at 12,000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (Thermo Fisher, NANODROP ONE). The effect of different amounts of ethanol and 200 μL of dichloromethane as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 6.

[0066] Table 6. Extraction effect of different amounts of ethanol and 200 μL dichloromethane on mRNA.

[0067] As shown in Table 6, when the sample volume is 100 μL and the dichloromethane volume is 200 μL, the ethanol volume is in the range of 100 μL to 150 μL, that is, when the volume ratio of sample-ethanol-dichloromethane is 1:1:2 to 1:1.5:2, good mRNA extraction results can be obtained.

[0068] (2) When the sample size is 100 μL and the ethanol volume is 100 μL, the effect of dichloromethane volume on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 portions. 100 μL of ethanol was added to each portion, followed by different amounts of dichloromethane (50 μL, 100 μL, 150 μL, 250 μL, and 300 μL). Each amount of dichloromethane was prepared in triplicate. The mixtures were mixed and centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (ThermoFisher, NANODROP ONE). The effect of different amounts of dichloromethane and 100 μL of ethanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 7.

[0069] Table 7. Extraction effect of different amounts of dichloromethane and 100 μL ethanol on mRNA.

[0070] As shown in Table 7, when the sample volume is 100 μL and the ethanol volume is 100 μL, the dichloromethane volume is in the range of 100 μL to 300 μL, that is, when the volume ratio of sample-ethanol-dichloromethane is 1:1:1 to 1:1:3, good mRNA extraction results can be obtained.

[0071] (3) When the sample size is 100 μL and the ethanol volume is 120 μL, the effect of dichloromethane volume on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 portions. 120 μL of ethanol was added to each portion, followed by different amounts of dichloromethane (50 μL, 100 μL, 150 μL, 250 μL and 300 μL). Each amount of dichloromethane was prepared in triplicate. The mixtures were mixed and centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (ThermoFisher, NANODROP ONE). The effect of different amounts of dichloromethane and 120 μL of ethanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 8.

[0072] Table 8. Extraction effect of different amounts of dichloromethane and 120 μL ethanol on mRNA.

[0073] As shown in Table 8, when the sample volume is 100 μL and the ethanol volume is 120 μL, the amount of dichloromethane used is in the range of 100 μL to 300 μL, that is, when the volume ratio of sample-ethanol-dichloromethane is 1:1.2:1 to 1:1.2:3, good mRNA extraction results can be obtained.

[0074] (4) When the sample size is 100 μL and the ethanol volume is 150 μL, the effect of dichloromethane dosage on mRNA extraction rate was investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 aliquots. 150 μL of ethanol was added to each aliquot, followed by different amounts of dichloromethane (50 μL, 100 μL, 150 μL, 250 μL, and 300 μL). Each amount of dichloromethane was prepared in triplicate. The aliquots were mixed and centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (ThermoFisher, NANODROP ONE). The effect of different amounts of dichloromethane and 150 μL of ethanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 9.

[0075] Table 9. Extraction effect of different amounts of dichloromethane and 150 μL ethanol on mRNA.

[0076] As shown in Table 9, when the sample volume is 100 μL and the ethanol volume is 150 μL, the amount of dichloromethane used is in the range of 100 μL to 300 μL, that is, when the volume ratio of sample-ethanol-dichloromethane is 1:1.5:1 to 1:1.5:3, good mRNA extraction results can be obtained.

[0077] Example 5 (1) When the sample size is 100 μL and the amount of dichloromethane is 200 μL, the effect of isopropanol dosage on mRNA extraction rate was investigated. Eighteen samples of mRNA-LNP prepared in Example 1 were prepared, each containing 100 μL of the sample. 200 μL of dichloromethane was added to each sample, followed by different amounts of isopropanol (50 μL, 80 μL, 100 μL, 120 μL, 150 μL, and 200 μL). Each isopropanol amount was prepared in triplicate. The samples were mixed, centrifuged at 12000 rpm for 2 min, and the supernatant was collected. The mRNA concentration was measured using a micro-ultraviolet spectrophotometer (ThermoFisher, NANODROP ONE). The effect of different amounts of isopropanol and 200 μL of dichloromethane as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 10.

[0078] Table 10. Extraction effect of different amounts of isopropanol and 200 μL of dichloromethane on mRNA.

[0079] As shown in Table 10, when the sample volume is 100 μL and the dichloromethane volume is 200 μL, the isopropanol volume is in the range of 100 μL to 150 μL, that is, when the volume ratio of sample-isopropanol-dichloromethane is 1:1:2 to 1:1.5:2, good mRNA extraction results can be obtained.

[0080] (2) When the sample size is 100 μL and the amount of isopropanol used is 100 μL, the effect of the amount of dichloromethane used on the mRNA extraction rate is investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 portions. 100 μL of isopropanol was added to each portion, followed by different amounts of dichloromethane (50 μL, 100 μL, 150 μL, 250 μL and 300 μL). Each amount of dichloromethane was prepared in triplicate. The mixtures were mixed and centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (ThermoFisher, NANODROP ONE). The effect of different amounts of dichloromethane and 100 μL of isopropanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 11.

[0081] Table 11. Extraction effect of different amounts of dichloromethane and 100 μL isopropanol on mRNA.

[0082] As shown in Table 11, when the sample volume is 100 μL and the amount of isopropanol is 100 μL, the amount of dichloromethane used is in the range of 100 μL to 300 μL, that is, when the volume ratio of sample-isopropanol-dichloromethane is 1:1:1 to 1:1:3, good mRNA extraction results can be obtained.

[0083] (3) When the sample size is 100 μL and the isopropanol volume is 120 μL, the effect of dichloromethane volume on mRNA extraction rate is investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 portions. 120 μL of isopropanol was added to each portion, followed by different amounts of dichloromethane (50 μL, 100 μL, 150 μL, 250 μL and 300 μL). Each amount of dichloromethane was prepared in triplicate. The mixtures were mixed and centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (ThermoFisher, NANODROP ONE). The effect of different amounts of dichloromethane and 120 μL of isopropanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 12.

[0084] Table 12. Extraction effect of different amounts of dichloromethane and 120 μL isopropanol on mRNA.

[0085] As shown in Table 12, when the sample volume is 100 μL and the isopropanol volume is 120 μL, the dichloromethane volume is in the range of 100 μL to 300 μL, that is, when the volume ratio of sample-isopropanol-dichloromethane is 1:1.2:1 to 1:1.2:3, good mRNA extraction results can be obtained.

[0086] (4) When the sample size is 100 μL and the isopropanol volume is 150 μL, the effect of dichloromethane volume on mRNA extraction rate is investigated. 100 μL of the mRNA-LNP sample prepared in Example 1 was measured and divided into 15 aliquots. 150 μL of isopropanol was added to each aliquot, followed by different amounts of dichloromethane (50 μL, 100 μL, 150 μL, 250 μL and 300 μL). Each amount of dichloromethane was prepared in triplicate. The aliquots were mixed and centrifuged at 12000 rpm for 2 min. The supernatant was collected and the mRNA concentration was measured using a micro-ultraviolet spectrophotometer (ThermoFisher, NANODROP ONE). The effect of different amounts of dichloromethane and 150 μL of isopropanol as the extraction solvent on the mRNA extraction efficiency was investigated. The results are shown in Table 13.

[0087] Table 13. Extraction effect of different amounts of dichloromethane and 150 μL isopropanol on mRNA.

[0088] As shown in Table 13, when the sample volume is 100 μL and the isopropanol volume is 150 μL, the dichloromethane volume should be in the range of 150 μL to 300 μL, that is, when the volume ratio of sample-isopropanol-dichloromethane is 1:1.5:1.5 to 1:1.5:3, good mRNA extraction results can be obtained.

[0089] Example 6 The integrity of the mRNA stock solution and the mRNA extracted with the extraction solvent of 120 μL methanol and 200 μL dichloromethane in Example 2 was detected using the Agilent RNA Kit (15NT) (Agilent, DNF-471-0500) and Fragment Analyzer. The results are as follows: Figure 1 and Figure 2 As shown in the figure. The test results indicate that the method provided by this invention for extracting mRNA from mRNA-LNP does not cause any damage to the mRNA during the extraction process. At the same concentration, the integrity of the original mRNA solution and the mRNA extracted from mRNA-LNP is consistent, both at 83.4%.

[0090] Example 7 The method for preparing mRNA-LNP samples in this embodiment includes the following steps: Weigh DODMA, DSPC, cholesterol, and mPEG-DMG-2K, dissolve them in ethanol to prepare a total lipid solution of 20 mg / mL, which is the ethanol phase; dissolve the mRNA stock solution in sodium citrate buffer to prepare a 0.33 mg / mL mRNA sodium acetate solution (25 mmol / L), which is the aqueous phase. Mix the two phases using a liquid chromatography pump, and then use PBS-sucrose solution for tangential ultrafiltration to obtain mRNA-LNP.

[0091] Specifically, the preparation method for mRNA-LNP samples includes the following steps: (1) Preparation of sodium citrate buffer: Weigh an appropriate amount of sodium citrate, dissolve it in water, and adjust the pH to 4.0 with citric acid to prepare a 50 mmol / L sodium citrate buffer (pH 4.0).

[0092] (2) Preparation of lipid solution: Weigh appropriate amounts of DODMA, DSPC, cholesterol and mPEG-DMG-2K in a molar ratio (50:10:38:2), dissolve in ethanol, bathe in a 40°C water bath until the lipids are completely dissolved, add ethanol to make up to a volume of 20 mg / mL lipid solution.

[0093] (3) Preparation of PBS-sucrose solution: Weigh appropriate amounts of potassium dihydrogen phosphate, disodium hydrogen phosphate, and sucrose, dissolve them in water, and prepare a 10 mmol / L PBS-150 mmol / L sucrose solution (pH 7.5).

[0094] (4) LNP self-assembly: 1) Add an appropriate amount of the mRNA stock solution with the code ZJHC001 to 50 mmol / L sodium citrate buffer, dilute with water to prepare an mRNA sodium citrate solution of about 0.33 mg / mL (25 mmol / L), and preheat in a water bath at 40°C. 2) Take an appropriate amount of lipid solution and preheat it in a water bath at 40℃; 3) Using a liquid phase pump, the lipid solution and the mRNA sodium citrate solution are rapidly mixed in a T-type flow rate ratio of 1:3 to form an LNP solution through self-assembly; 4) After mixing, add 4 times the volume of PBS-sucrose solution to the LNP solution formed in step 3).

[0095] (5) Ultrafiltration: Using PBS-sucrose solution as the replacement solution, the hollow fiber column was used for tangential flow ultrafiltration 5 times to obtain mRNA-LNP.

[0096] The obtained mRNA-LNP contained approximately 3.6 mg / mL of total lipids and approximately 180 μg / mL of mRNA, with a drug-to-lipid ratio of 1:20.

[0097] Example 8 The method for preparing mRNA-LNP samples in this embodiment includes the following steps: Weigh SM-102, DSPC, cholesterol, and mPEG-DMG-2K, dissolve them in ethanol, and prepare a total lipid solution of 20 mg / mL as the organic phase; dissolve the mRNA stock solution in sodium acetate buffer to prepare a 0.33 mg / mL mRNA sodium acetate solution (25 mmol / L) as the aqueous phase; mix the two phases using a liquid phase pump, and then use Tris-sucrose solution for tangential flow ultrafiltration to obtain mRNA-LNP.

[0098] Specifically, the preparation method for mRNA-LNP samples includes the following steps: (1) Preparation of sodium acetate buffer: Weigh an appropriate amount of sodium acetate, dissolve it in water, and adjust the pH to 5.2 with acetic acid to prepare a 50 mmol / L sodium acetate buffer (pH 5.2).

[0099] (2) Preparation of lipid solution: Weigh appropriate amounts of SM-102, DSPC, cholesterol and mPEG-DMG-2K in a molar ratio (50:10:38:2), dissolve in ethanol, bathe in a 40℃ water bath until the lipids are completely dissolved, add ethanol to make up to a volume of 20 mg / mL lipid solution.

[0100] (3) Preparation of Tris-sucrose solution: Weigh appropriate amounts of Tris and sucrose, dissolve them in water, adjust the pH to 7.5 with hydrochloric acid, and prepare a Tris-sucrose solution of 25 mmol / L Tris - 300 mmol / L sucrose (pH 7.5).

[0101] (4) LNP self-assembly: 1) Add an appropriate amount of the mRNA stock solution with the substitution number ZJHC001 to 50 mmol / L sodium acetate buffer, dilute with water to prepare an mRNA sodium acetate solution of about 0.33 mg / mL (25 mmol / L), and preheat in a water bath at 40℃. 2) Take an appropriate amount of lipid solution and preheat it in a water bath at 40℃; 3) Using a liquid phase pump, the lipid solution and the sodium acetate solution of mRNA are rapidly mixed in a T-type flow rate ratio of 1:3 to form an LNP solution through self-assembly; 4) After mixing, add 4 times the volume of Tris-sucrose solution to the LNP solution formed in step 3).

[0102] (5) Ultrafiltration: Using Tris-sucrose solution as the replacement solution, the hollow fiber column was used for tangential flow ultrafiltration 5 times to obtain mRNA-LNP.

[0103] The obtained mRNA-LNP contained approximately 3.6 mg / mL of total lipids and approximately 180 μg / mL of mRNA, with a drug-to-lipid ratio of 1:20.

[0104] Example 9 The total lipid concentration in the prepared mRNA-LNP was approximately 1.8 mg / mL, and the drug-to-lipid ratio was 1:10. The rest was the same as in Example 7.

[0105] Example 10 The total lipid concentration in the prepared mRNA-LNP was approximately 7.2 mg / mL, and the drug-to-lipid ratio was 1:40. The rest was the same as in Example 7.

[0106] Example 11 Methanol and dichloromethane were used as reagents to extract mRNA from the mRNA-LNP samples prepared in Examples 7-10. Extraction method: 100 μL of sample was taken, 120 μL of methanol was added, followed by 200 μL of dichloromethane. The mixture was stirred, centrifuged at 12000 rpm for 2 min, and the supernatant was used to determine the mRNA concentration. The extraction results are shown in Tables 14-17.

[0107] Table 14. mRNA extraction results of the mRNA-LNP samples prepared in Example 7

[0108] Table 15. mRNA extraction results of the mRNA-LNP samples prepared in Example 8.

[0109] Table 16. mRNA extraction results of the mRNA-LNP samples prepared in Example 9

[0110] Table 17 mRNA extraction results of the mRNA-LNP samples prepared in Example 10

[0111] Experimental Example 1: Methodological Validation Based on the results of Examples 1, 3 and 4, the combination of methanol-dichloromethane, ethanol-dichloromethane and isopropanol-dichloromethane can achieve good mRNA extraction results within a certain proportion range. The median value of reagent dosage for each combination was selected as the extraction method for methodological validation.

[0112] 1. Extraction of mRNA with methanol-dichloromethane Extraction method: Take 100 μL of the mRNA-LNP sample prepared in Example 1, add 120 μL of methanol, then add 200 μL of dichloromethane, mix well, centrifuge at 12000 rpm for 2 min, and take the supernatant to determine the mRNA concentration.

[0113] (1) Accuracy The theoretical concentration of mRNA in the mRNA-LNP sample is 180 μg / mL. Solutions with concentrations of 90 μg / mL, 180 μg / mL, and 270 μg / mL were designed as 50%, 100%, and 150% accuracy solutions, respectively.

[0114] Take 10 μL of mRNA (ZJHC001) stock solution with a concentration of 1650 μg / mL, add 173 μL of blank LNP (without drug loading, other components are the same as in Example 1), mix well, and prepare three parallel aliquots as 50% accuracy solutions.

[0115] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 83 μL of blank LNP, mix well, and prepare three parallel aliquots as 100% accuracy solutions.

[0116] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 51 μL of blank LNP, mix well, and prepare three parallel aliquots as 150% accuracy solutions.

[0117] mRNA was extracted using the method described above, and the results are shown in Table 18.

[0118] Table 18 Results of methanol-dichloromethane extraction accuracy

[0119] As shown in Table 18, the method for extracting mRNA in this invention has a mRNA recovery rate of 96.7% to 105.0% and good accuracy.

[0120] (2) Repeatability Six mRNA-LNP samples were taken and mRNA was extracted according to the above extraction method. The extraction results are shown in Table 19.

[0121] Table 19 Repeatability Results of Methanol-Dichloromethane

[0122] As shown in Table 19, the method for extracting mRNA in this invention has an mRNA recovery rate of 93.9% to 102.8%, an average recovery rate of 99.1%, an RSD of 3.0%, and good reproducibility.

[0123] (3) Intermediate precision Take a sample of mRNA-LNP and extract mRNA according to the above extraction method. The extraction results are shown in Tables 20-21.

[0124] Table 20 Intermediate Precision Results for Methanol-Dichloromethane

[0125] Table 21 Extraction results of 12 methanol-dichloromethane test samples

[0126] As shown in Table 20, the mRNA extraction method of the present invention has an mRNA extraction recovery rate of 99.4%~103.3%, an average recovery rate of 101.6%, and an RSD of 1.5%; as shown in Table 21, the mRNA extraction method of the present invention has an mRNA extraction recovery rate of 93.9%~103.3%, an average recovery rate of 100.3%, and an RSD of 2.6%, indicating good intermediate precision.

[0127] 2. Extraction of mRNA using ethanol-dichloromethane Extraction method: Take 100 μL of the mRNA-LNP sample prepared in Example 1, add 120 μL of ethanol, then add 200 μL of dichloromethane, mix well, centrifuge at 12000 rpm for 2 min, and take the supernatant to determine the mRNA concentration.

[0128] (1) Accuracy The theoretical concentration of mRNA in the mRNA-LNP sample is 180 μg / mL. Solutions with concentrations of 90 μg / mL, 180 μg / mL, and 270 μg / mL were designed as 50%, 100%, and 150% accuracy solutions, respectively.

[0129] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 173 μL of blank LNP (without drug loading, all other components are the same), mix well, and prepare three parallel aliquots as 50% accuracy solutions.

[0130] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 83 μL of blank LNP, mix well, and prepare three parallel aliquots as 100% accuracy solutions.

[0131] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 51 μL of blank LNP, mix well, and prepare three parallel aliquots as 150% accuracy solutions.

[0132] mRNA was extracted using the method described above, and the results are shown in Table 22.

[0133] Table 22 Results of Ethanol-Dichloromethane Extraction Accuracy

[0134] As shown in Table 22, the method for extracting mRNA in this invention has a mRNA recovery rate of 98.9% to 107.0% and good accuracy.

[0135] (2) Repeatability Six mRNA-LNP samples were taken and mRNA was extracted according to the above extraction method. The extraction results are shown in Table 23.

[0136] Table 23 Repeatability Results of Ethanol-Dichloromethane

[0137] As shown in Table 23, the method for extracting mRNA in this invention has an mRNA recovery rate of 96.7% to 103.9%, an average recovery rate of 100.5%, an RSD of 3.0%, and good reproducibility.

[0138] (3) Intermediate precision Take a sample of mRNA-LNP and extract mRNA according to the above extraction method. The extraction results are shown in Tables 24 and 25.

[0139] Table 24 Intermediate Precision Results for Ethanol-Dichloromethane

[0140] Table 25 Extraction results of 12 test samples of ethanol-dichloromethane

[0141] As shown in Table 24, the mRNA extraction method of the present invention has an mRNA extraction recovery rate of 96.7%~106.1%, an average recovery rate of 102.2%, and an RSD of 3.1%; as shown in Table 25, the mRNA extraction method of the present invention has an mRNA extraction recovery rate of 96.7%~106.1%, an average recovery rate of 101.3%, and an RSD of 3.1%, indicating good intermediate precision.

[0142] 3. Extraction of mRNA using isopropanol-dichloromethane Extraction method: Take 100 μL of the mRNA-LNP sample prepared in Example 1, add 120 μL of isopropanol, then add 200 μL of dichloromethane, mix well, centrifuge at 12000 rpm for 2 min, and take the supernatant to determine the mRNA concentration.

[0143] (1) Accuracy The theoretical concentration of mRNA in the mRNA-LNP sample is 180 μg / mL. Solutions with concentrations of 90 μg / mL, 180 μg / mL, and 270 μg / mL were designed as 50%, 100%, and 150% accuracy solutions, respectively.

[0144] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 173 μL of blank LNP (without drug loading, all other components are the same), mix well, and prepare three parallel aliquots as 50% accuracy solutions.

[0145] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 83 μL of blank LNP, mix well, and prepare three parallel aliquots as 100% accuracy solutions.

[0146] Take 10 μL of mRNA stock solution with a concentration of 1650 μg / mL, add 51 μL of blank LNP, mix well, and prepare three parallel aliquots as 150% accuracy solutions.

[0147] mRNA was extracted using the method described above, and the results are shown in Table 26.

[0148] Table 26. Results of Isopropanol-Dichloromethane Extraction Accuracy

[0149] As shown in Table 26, the method for extracting mRNA in this invention has a mRNA recovery rate of 93.3% to 105.2% and good accuracy.

[0150] (2) Repeatability Six mRNA-LNP samples were taken and mRNA was extracted according to the above extraction method. The extraction results are shown in Table 27.

[0151] Table 27 Repeatability Results of Isopropanol-Dichloromethane

[0152] As shown in Table 27, the method for extracting mRNA in this invention has an mRNA recovery rate of 97.2% to 108.3%, an average recovery rate of 101.7%, an RSD of 4.1%, and good reproducibility.

[0153] (3) Intermediate precision Take a sample of mRNA-LNP and extract mRNA according to the above extraction method. The extraction results are shown in Tables 28 and 29.

[0154] Table 28. Precise intermediate results of isopropanol-dichloromethane

[0155] Table 29 Extraction results of 12 samples of isopropanol-dichloromethane

[0156] As shown in Table 28, the mRNA extraction method of the present invention has an mRNA extraction recovery rate of 98.9%~07.2%, an average recovery rate of 103.5%, and an RSD of 3.1%; as shown in Table 29, the mRNA extraction method of the present invention has an mRNA extraction recovery rate of 97.2%~108.3%, an average recovery rate of 102.6%, and an RSD of 3.6%, indicating good intermediate precision.

[0157] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for rapidly extracting mRNA from mRNA-LNP, characterized in that, Includes the following steps: A polar organic solvent and a non-polar organic solvent are added to the mRNA-LNP sample to obtain a mixture; After the mixture has been left to stand or centrifuged, the supernatant is collected to obtain the mRNA solution. The polar organic solvent is selected from at least one of alcohols, ketones and amides, and the nonpolar organic solvent is selected from at least one of alkanes, halogenated hydrocarbons and esters.

2. The method according to claim 1, characterized in that, The polar organic solvent is selected from at least one of alcohol-based organic solvents, and the non-polar organic solvent is selected from at least one of halohydrocarbon-based organic solvents.

3. The method according to claim 2, characterized in that, The polar organic solvent is selected from at least one of methanol, ethanol, isopropanol and butanol, and the nonpolar organic solvent is selected from dichloromethane, trichloromethane or a combination thereof.

4. The method according to claim 3, characterized in that, The total lipid concentration in the mRNA-LNP sample was 1.8–36 mg / mL.

5. The method according to claim 4, characterized in that, In the mRNA-LNP sample, the mass ratio of mRNA to total lipids is 1:5 to 1:

40.

6. The method according to claim 5, characterized in that, The volume ratio of the mRNA-LNP sample, polar organic solvent, and non-polar organic solvent is 1:(1~1.5):(0.8~6).

7. The method according to any one of claims 1 to 6, characterized in that, The lipid components of the mRNA-LNP sample include ionizable lipids, neutral lipids, and polyethylene glycol lipids, or are composed of ionizable lipids, neutral lipids, and polyethylene glycol lipids.

8. The method according to claim 7, characterized in that, The neutral lipids include phosphate-containing neutral lipids and sterol lipids.

9. The method according to claim 7, characterized in that, The ionizable lipids are selected from one or more of DODMA, ALC-0159, A066, L-319, DODAP, C12-200, 5A2-SC8, 306Oi10, 306-O12B-3, Moderna Lipid5, AcuitasA9, ALC-0315, SM-102, DLin-MC3-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, and DLinDMA.

10. The method according to claim 8, characterized in that, The phosphate-containing neutral lipids are selected from one or more of the following: distearylphosphatidylcholine, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dioleoylphosphatidylethanolamine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoylphosphatidylcholine, diarachidoylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, distearylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylethanolamine, 1,2-distearyl-sn-glycerol-3-phosphatidylglycerol, and 1-hexadecane-2-(cis-9-octadecenoyl)-sn-glycerol-3-phosphocholine.

11. The method according to claim 8, characterized in that, The sterol lipids are selected from one or more of β-sitosterol, stigmasterol, campesterol, cholesterol, and sterol derivatives.

12. The method according to claim 7, characterized in that, The polyethylene glycol lipid is selected from one or more of PEG-docoalkylacetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-phospholipid, PEG-ceramide, DMG-PEG, DSPE-PEG and DSG-PEG.

13. The method according to any one of claims 8 to 12, characterized in that, The lipid component of the mRNA-LNP sample also includes permanent cationic lipids.

14. The method according to claim 13, characterized in that, The permanent cationic lipid is selected from one or more of DOTAP, DOTMA, DDBA, DMRIE, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOSPA, DORIE, DODAB, DOIC, DMEPC, DOGS, DIMRI, DC-6-14, CLIP 1, DORIE, DOSPA, CLIP 6, and CLIP 9.

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