Pretreatment method of Oligo-LNP sample

Abstract

The invention discloses a pretreatment method of an Oligo-LNP sample, which comprises the following steps: adding an extracting agent into the Oligo-LNP sample, standing or centrifuging, and taking an upper layer solution to obtain an Oligo solution; wherein the extraction agent is composed of a first organic reagent and a second organic reagent; the first organic reagent is selected from an alcohol organic solvent; the second organic reagent is selected from halogenated hydrocarbon organic solvents. According to the pretreatment method of the Oligo-LNP sample, provided by the invention, Oligo and related impurities thereof can be simply, quickly and efficiently released from an Oligo-LNP preparation, and an Oligo solution containing high-concentration Oligo and related impurities thereof is obtained. The Oligo solution obtained by the pretreatment method provided by the invention can be used for related substance inspection, impurity research, sequence identification analysis and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical preparation analysis, and relates to a pretreatment method for Oligo-LNP samples. BACKGROUND

[0002] Oligonucleotide drugs are a kind of drugs composed of 12-30 oligonucleotide single chains or double chains artificially synthesized. Oligonucleotide drugs are a new type of drugs after small molecule drugs and protein drugs, and are one of the hotspots in drug development in recent years. Oligonucleotides have low extracellular stability and are easily degraded by nucleases, and have the factors of large molecular weight, strong hydrophilicity and high negative charge, which show poor drugability, and the pharmacokinetic characteristics are poor, and it is not easy to penetrate the lipid bilayer and the blood-brain barrier, and usually needs to be improved by chemical modification or drug delivery system. The lipid nanoparticle (LNP) delivery system is one of the common oligonucleotide delivery systems in clinical use.

[0003] After the Oligo-LNP preparation product is prepared, the Oligo-LNP needs to be pretreated to release the Oligo from the LNP before analysis such as related substance examination and sequence identification analysis, that is, demulsification is needed. Usually, 0.1%-2% triton X-100 or other surfactants are selected as demulsifiers. When the surfactant is selected as the demulsifier for dilution, the surfactant is easy to foam and has a certain turbidity itself, and it is difficult to observe whether the LNP is completely dissolved with the naked eye; and the surfactant is also strongly retained in the chromatographic column during subsequent analysis such as related substance examination, which causes the pressure of the chromatographic column to rise and even causes the chromatographic column to be blocked. In addition, the concentration of the Oligo-LNP preparation product is generally not more than 5 mg / mL, and if a large dilution factor is needed, the sensitivity cannot meet the requirements. If an organic reagent such as methanol, isopropanol, acetonitrile or the like is directly used for dilution and demulsification, the Oligo will have a serious solvation effect and will not be retained by the chromatographic column, so that the related substance examination cannot be performed. SUMMARY

[0004] The purpose of the present application is to provide a pretreatment method for Oligo-LNP samples to solve at least one of the above technical problems.

[0005] According to one aspect of the present application, a pretreatment method for Oligo-LNP samples is provided, comprising the following steps: An extractant is added to the Oligo-LNP sample, and the mixture is left to stand or centrifuged, and the upper solution is taken, to obtain an Oligo solution; wherein the extractant is composed of a first organic reagent and a second organic reagent.

[0006] In some embodiments, the first organic reagent is selected from an alcohol organic solvent, and the second organic reagent is selected from a halogenated hydrocarbon organic solvent.

[0007] The Oligo-LNP sample contains a large amount of lipid components, and a single solvent cannot meet the dissolution requirements of the various lipid components in the Oligo-LNP. Through a large amount of research, it is found that the alcohol polar protic solvent, the medium aprotic solvent or the weak polar protic solvent, and the water, in a certain proportion, can be combined into a mixed reagent most suitable for the dissolution of the various lipid components in the Oligo-LNP; when the alcohol polar protic solvent, the medium aprotic solvent or the weak polar protic solvent, and the Oligo-LNP sample are mixed, the differences in solubility and density between the three will cause the solution to stratify. Among them, the lower layer mixed solution is mainly composed of the medium aprotic solvent or the weak polar protic solvent (such as chloroform, dichloromethane, etc.) with a higher density, and also dissolves the alcohol polar protic solvent (such as methanol, ethanol, isopropanol, etc.) and the water in the Oligo-LNP sample. By adjusting the volume ratio of the alcohol polar protic solvent, the medium aprotic solvent or the weak polar protic solvent, and the Oligo-LNP sample, a lower layer mixed solution very beneficial to the dissolution of the lipid components can be obtained, and the lipid components in the Oligo-LNP sample can be extracted into the lower layer solution. The upper layer solution is mainly composed of water, mixed with a small amount of alcohol polar protic solvent and medium aprotic solvent or weak polar protic solvent, and the water-soluble components in the Oligo-LNP are naturally extracted into the upper layer mixed solution. In addition to Oligo, the water-soluble components in the Oligo-LNP also include Tri, PBS buffer, sucrose, etc., which can maintain a good solution pH environment (7-8) in the upper layer and also provide a certain ionic strength. In this neutral to weakly alkaline solution environment with a certain ionic strength, the phosphate groups in Oligo are fully dissociated, the intermolecular electrostatic repulsion is strong, and the hydrogen bond interaction with water molecules is stable, which is conducive to the extraction of Oligo into the water layer. In addition, the PBS buffer is usually low in concentration, and sucrose is a neutral molecule, which usually has no effect on the extraction of Oligo.

[0008] The pretreatment method of the Oligo-LNP sample provided by the application can simply, quickly and efficiently release Oligo and its related impurities from the Oligo-LNP preparation. After mixing the Oligo-LNP sample and the extractant, the supernatant is removed by centrifugation to obtain an Oligo solution containing high concentrations of Oligo and its related impurities. The Oligo solution obtained by the pretreatment method provided by the application can be used for related substance inspection, impurity research and sequence identification analysis, etc. The pretreatment method provided by the application can well assist in the research of the prescription process of Oligo-LNP preparation and the quality research of Oligo-LNP preparation products.

[0009] In some embodiments, the first organic reagent can be selected from at least one of methanol, ethanol, isopropanol, and butanol.

[0010] In some embodiments, the second organic reagent can be selected from dichloromethane, trichloromethane, or a combination thereof.

[0011] In some embodiments, the concentration of total lipids in the Oligo-LNP sample is 1.6-32 mg / mL.

[0012] In some embodiments, the mass ratio of Oligo to total lipids (drug-lipid ratio) in the Oligo-LNP sample is 1:5-1:40. Preferably, the drug-lipid ratio is 1:5, 1:8, 1:10, 1:15, and 1:20.

[0013] In some embodiments, the concentration of Oligo in the Oligo-LNP sample is no more than 100 mg / mL.

[0014] In some embodiments, the volume ratio of the Oligo-LNP sample, the first organic reagent, and the second organic reagent can be 1:(0.5-2.5):(1-3).

[0015] In some embodiments, when the extractant consists of methanol and dichloromethane, the volume ratio of the Oligo-LNP sample, methanol, and dichloromethane can be 1:(1-1.5):(1-3). Preferably, when the amount of dichloromethane is 100 μL, the volume ratio of the Oligo-LNP sample, methanol, and dichloromethane is 1:1:1, and Oligo can obtain a higher extraction rate; when the amount of dichloromethane is 200 μL, the volume ratio of the Oligo-LNP sample, methanol, and dichloromethane is in the range of 1:1:2-1:1.5:2, and Oligo can obtain a higher extraction rate; when the amount of dichloromethane is 300 μL, the volume ratio of the Oligo-LNP sample, methanol, and dichloromethane is in the range of 1:1:3-1:1.5:3, and Oligo can obtain a higher extraction rate.

[0016] In some embodiments, when the extraction agent consists of ethanol and dichloromethane, the volume ratio of Oligo-LNP sample, ethanol, dichloromethane can be 1: (0.5-2.5): (1-3). Preferably, when the amount of dichloromethane is 100 μL, the volume ratio of Oligo-LNP sample, ethanol, dichloromethane is in the range of 1:0.5:1-1:1.5:1, and Oligo can obtain a higher extraction rate; when the amount of dichloromethane is 200 μL, the volume ratio of Oligo-LNP sample, ethanol, dichloromethane is in the range of 1:0.5:2-1:2:2, and Oligo can obtain a higher extraction rate; when the amount of dichloromethane is 300 μL, the volume ratio of Oligo-LNP sample, ethanol, dichloromethane is in the range of 1:0.5:3-1:2.5:3, and Oligo can obtain a higher extraction rate.

[0017] In some embodiments, when the extraction agent consists of isopropanol and dichloromethane, the volume ratio of Oligo-LNP sample, isopropanol, dichloromethane can be 1: (0.5-2.5): (1-3). Preferably, when the amount of dichloromethane is 100 μL, the volume ratio of Oligo-LNP sample, isopropanol, dichloromethane is in the range of 1:0.5:1-1:1.5:1, and Oligo can obtain a higher extraction rate; when the amount of dichloromethane is 200 μL, the volume ratio of Oligo-LNP sample, isopropanol, dichloromethane is in the range of 1:0.5:2-1:2:2, and Oligo can obtain a higher extraction rate; when the amount of dichloromethane is 300 μL, the volume ratio of Oligo-LNP sample, isopropanol, dichloromethane is in the range of 1:1:3-1:2.5:3, and Oligo can obtain a higher extraction rate.

[0018] In some embodiments, the centrifugal speed can be (0-12000) rpm, and the centrifugal time can be (1-10) min. The centrifugal time and centrifugal speed have no significant effect on the extraction effect of Oligo.

[0019] In some embodiments, the centrifugal speed can be 12000 rpm, and the centrifugal time can be 2 min. In this way, not only time can be effectively saved, but also a higher Oligo extraction rate can be obtained.

[0020] In some embodiments, the lipid component of the Oligo-LNP sample includes an ionizable cationic lipid, a phosphoric acid group-containing neutral lipid, a sterol lipid, and a polymer-conjugated lipid.

[0021] In some embodiments, the ionizable cationic lipid can be selected from one or more of DODMA (1,2-dioleyloxy-3-dimethylaminopropane, 1,2-Dioleyloxy-3-dimethylamino-propane, CAS: 104162-47-2), ALC-0315 (CAS: 2036272-55-4), YK-201, PL40, DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4- (dimethylamino)butanoate), SM-102 (also known as HUO, Moderna Lipid H, 1- octylnonyl 58-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate), VN2C1, C12-200 (CAS: 1220890-25-4), Lipid HTO12, TNT-b10, Lipid 20b, C14-4, 5A2-SC8 (CAS: 1857341-90-2), CKK-E12, DLin-KC2-DMA, 306Oi10 (CAS: 2322290-93-5), TCL053, 306-O12B (CAS: 2566523-06-4), Genevant CL1, L319, OF-C4-Deg-Lin, PPZ-A10, Ionizable lipid-1, OF-02, Lipid 331, OF-Deg-Lin, ATX-002, 244cis, 113-N16B, Ionizable lipid-2, AA3-Dlin, C10-200, 113-O12B, A12-Iso5-2DC18, 4A3-Cit, RCB-02-4-8, Lipid AX4, Lipid DIM1, 9322-O16B, Al-28, 306Oi9-cis2.

[0022] In some embodiments, the phosphogroup-containing neutral lipid can be selected from one or more of distearoylphosphatidylcholine (DSPC), egg yolk lecithin (EPC), soybean phospholipid, hydrogenated soybean phospholipid (HSPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoylphosphatidylcholine (DOPC), diarachidoylphosphatidylcholine (DAPC), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG), 1-hexadecyl 2-(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine (POPC).

[0023] In some embodiments, the sterol lipid can be selected from one or more of beta-sitosterol, stigmasterol, campesterol, cholesterol, and sterol derivatives.

[0024] In some embodiments, the polymer-conjugated lipid can be selected from one or more of polyethylene glycol-conjugated lipids. Among them, the polyethylene glycol-conjugated lipids include, but are not limited to, one or more of PEG-dodecanoylacetamide, PEG-myristoyldiglyceride, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-phospholipid, PEG-ceramide, DMG-PEG, DSPE-PEG, and DSG-PEG.

[0025] In some embodiments, the lipid component of the Oligo-LNP sample can further include a permanent cationic lipid.

[0026] The extraction system of the present application is used to extract Oligo from Oligo-LNP containing permanent cationic lipids. The effect is more significant when the LNP encounters a mixed solvent composed of an alcohol polar protic solvent, a medium polar aprotic solvent or a weak polar protic solvent and water in a certain proportion. The LNP lipid bilayer gradually dissolves, and the mixed solvent further enters the core site where the Oligo is combined with the cationic lipid. If the LNP contains a permanent cationic lipid, because of its hydrophilic group N + The O-H (dipole negative end) of the alcohol polar protic solvent (such as methanol) or the O-H (dipole negative end) of water in the mixed solvent can form a strong ion-dipole interaction with the permanent cationic lipid combined with the Oligo, which will quickly and competitively capture the permanent cationic lipid combined with the Oligo, not only promoting the dissolution of the permanent cationic lipid, but also promoting the release of the Oligo to the water phase, thereby effectively improving the extraction rate of the Oligo.

[0027] In some embodiments, the permanent cationic lipid can be selected from one or more of 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.

[0028] In some embodiments, the lipid component of the Oligo-LNP sample comprises or consists of an ionizable cationic lipid, a phosphate group-containing neutral lipid, a sterol lipid, and a polyethylene glycol-conjugated lipid. In terms of molar ratio (mol%) of total lipids, the ionizable cationic lipid: the phosphate group-containing neutral lipid: the sterol lipid: the polyethylene glycol-conjugated lipid is (30-70):(10-40):(20-50):(1-20).

[0029] In some embodiments, the lipid component of the Oligo-LNP sample consists of DODMA, DOPC, Cholesterol, DMG-PEG-2K, in a molar ratio of 45:15:38:2.

[0030] In some embodiments, the lipid component of the Oligo-LNP sample consists of SM-102, DOPC, Cholesterol, DMG-PEG-2K, in a molar ratio of 45:15:38:2.

[0031] In some embodiments, the lipid component of the Oligo-LNP sample comprises or consists of a permanent cationic lipid, an ionizable cationic lipid, a phosphate group-containing neutral lipid, a sterol lipid, and a polyethylene glycol-conjugated lipid. In terms of molar ratio (mol%) of total lipids, the permanent cationic lipid: the ionizable cationic lipid: the phosphate group-containing neutral lipid: the sterol lipid: the polyethylene glycol-conjugated lipid is (1-20):(30-50):(10-40):(10-40):(1-20).

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

[0033] In some embodiments, the lipid component of the Oligo-LNP sample consists of DOTAP, DODMA, DSPC, cholesterol, DMG-PEG-2K in a molar ratio of 15:25:20:38.5:1.5.

[0034] In some embodiments, the lipid component of the Oligo-LNP sample consists of DOTAP, DODMA, DOPC, cholesterol, DMG-PEG in a molar ratio of 5:40:30:20:5.

[0035] In some embodiments, the lipid component of the Oligo-LNP sample consists of DOTAP, DODMA, DOPC, cholesterol, DMG-PEG in a molar ratio of 5:40:27.5:20:7.5.

[0036] The beneficial effects of the present application include: (1) The pre-treatment method of the Oligo-LNP sample provided by the present application does not use surfactants, which can avoid damage to the chromatographic column during subsequent further analysis, prolong the service life of the chromatographic column, and obtain a clear and transparent solution that can be determined by the naked eye whether the Oligo-LNP sample is completely dissolved during the treatment process, which is simple and convenient.

[0037] (2) The pre-treatment method provided by the present application only needs to perform simple reagent addition and centrifugation operation, which is simple, fast and efficient.

[0038] (3) The Oligo sample obtained by the pre-treatment method provided by the present application has high concentration, which can still be diluted with water under the condition of ensuring sensitivity, and the organic solvent content of the sample after dilution is low, which avoids the phenomenon that the sample cannot be retained in the chromatographic column due to the solvation effect caused by the large amount of organic solvent in the sample caused by the direct use of organic solvent demulsification.

[0039] (4) The pre-treatment method provided by the present application is suitable for the pre-treatment of Oligo in Oligo-LNP samples with different lipid components, has wide application range, and is especially suitable for Oligo-LNP samples containing permanent cationic lipids. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figures 1-5 The chromatograms of blank control, API control, test sample 1 in pre-treatment method 1, test sample 3 in pre-treatment method 2 and test sample 2 in pre-treatment method 3 in sequence. DETAILED DESCRIPTION

[0041] The application will be further described in connection with the following embodiments. The embodiments are only used for explanation and do not limit the application in any way. If not otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be commercially available; the experimental methods not specified in the embodiments are usually carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturers.

[0042] In the embodiments of the application, the nucleotide sequence of the exemplary Oligo for experimental research, designated as ZJHC00A, is 5'-TCGAACGTTCGAACGTTCGAACGTAT-3'.

[0043] Embodiment 1 The preparation method of the Oligo-LNP sample includes the following steps: DODMA, DSPC, cholesterol, mPEG-DMG-2K, and DOTAP were weighed, dissolved in ethanol, and prepared into a total lipid solution with a total lipid concentration of 16 mg / mL as the ethanol phase; Oligo was dissolved in a sodium citrate buffer to prepare an Oligo sodium citrate solution (25 mol / L) with a concentration of 0.33 mg / mL as the water phase; the two phases were mixed by a liquid phase pump, and then ultrafiltrated by a tangential flow with a PBS-sucrose solution to obtain Oligo-LNP.

[0044] Specifically, the preparation method of the Oligo-LNP sample can include the following steps: (1) Preparation of a sodium citrate buffer: an appropriate amount of sodium citrate was weighed, dissolved in water, and adjusted to pH 4.0 with citric acid to prepare a 50 mmol / L sodium citrate buffer (pH 4.0).

[0045] (2) Preparation of a lipid solution: DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG-2K were weighed in a molar ratio of 15:25:20:38.5:1.5, dissolved in ethanol, and dissolved in water at 60°C until the lipids were completely dissolved to prepare a lipid solution with a total lipid concentration of 16 mg / mL.

[0046] (3) Preparation of a PBS-sucrose solution: potassium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride, and sucrose were weighed, dissolved in water, and prepared into a 10 mmol / L PBS-150 mmol / L sucrose solution (pH 7.5).

[0047] (4) LNP self-assembly: 1) Take the Oligo with the code ZJHC00A, add it to the 50 mmol / L sodium citrate buffer, dilute with water, and prepare an Oligo sodium citrate solution (25 mmol / L) of about 0.33 mg / mL, and preheat in a 40°C water bath; 2) Take the appropriate amount of lipid solution and preheat in a 40°C water bath; 3) Use a liquid pump to mix the lipid solution and the Oligo sodium citrate solution at a flow rate ratio of 1:3 Y type, and form an LNP solution by self-assembly; 4) After mixing, add 4 times the volume of the LNP solution formed in step 3) to the PBS-sucrose solution.

[0048] (5) Ultrafiltration: use a hollow fiber column for tangential flow ultrafiltration 5 times with PBS-sucrose solution as the displacement solution to obtain Oligo-LNP.

[0049] Among the obtained Oligo-LNP, the total lipid concentration is about 16 mg / mL, the Oligo concentration is about 1.6 mg / mL, and the drug-lipid ratio is 1:10.

[0050] Example 2 (1) Investigation of the extraction effect of extractants composed of 100 μL dichloromethane and different amounts of methanol Take 100 μL of the Oligo-LNP sample prepared in Example 1, a total of 12 portions, add 100 μL of dichloromethane, and then add different amounts of methanol (50 μL, 100 μL, 150 μL, 200 μL), each methanol amount is prepared in triplicate, mix well, centrifuge at 12000 rpm for 2 min, take the supernatant, measure its volume, and use a micro UV spectrophotometer (ThermoFisher, NANODROP ONE) to measure the Oligo concentration, investigate the extraction effect of extractants composed of different amounts of methanol and 100 μL dichloromethane on Oligo, and the results are shown in Table 1.

[0051] Table 1 Extraction effect of extractants composed of 100 μL dichloromethane and different amounts of methanol on Oligo

[0052] As shown in Table 1, when the sample is 100 μL, the dichloromethane amount is 100 μL, and the sample-methanol-dichloromethane volume ratio is 1:1:1, the Oligo can obtain good extraction effect.

[0053] (2) Investigation of the extraction effect of extractants composed of 200 μL dichloromethane and different amounts of methanol Take 100 μL of the Oligo-LNP sample prepared in Example 1, a total of 18 portions, add dichloromethane 200 μL respectively, and then add different amounts of methanol (100 μL, 150 μL, 200 μL, 250 μL, 300 μL and 400 μL) respectively. Each amount of methanol is prepared in triplicate. Mix well, centrifuge at 12000 rpm for 2 min, take the supernatant, measure its volume, and use a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE) to measure the Oligo concentration. The effect of different amounts of methanol and 200 μL of dichloromethane on the extraction of Oligo is investigated, and the results are shown in Table 2.

[0054] Table 2 Extraction effect of Oligo by different amounts of methanol and 200 μL of dichloromethane

[0055] As shown in Table 2, when the sample is 100 μL and the amount of dichloromethane is 200 μL, the amount of methanol used is in the range of 100 μL to 150 μL, i.e. the volume ratio of sample-methanol-dichloromethane is 1:1:2 to 1:1.5:2, and Oligo can obtain good extraction effect.

[0056] (3) Investigation of the extraction effect of 300 μL of dichloromethane and different amounts of methanol Take 100 μL of the Oligo-LNP sample prepared in Example 1, a total of 18 portions, add dichloromethane 300 μL respectively, and then add different amounts of methanol (50 μL, 100 μL, 150 μL, 200 μL, 250 μL and 300 μL) respectively. Each amount of methanol is prepared in triplicate. Mix well, centrifuge at 12000 rpm for 2 min, take the supernatant, measure its volume, and use a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE) to measure the Oligo concentration. The effect of different amounts of methanol and 300 μL of dichloromethane on the extraction of Oligo is investigated, and the results are shown in Table 3.

[0057] Table 3 Extraction effect of Oligo by different amounts of methanol and 300 μL of dichloromethane

[0058] As shown in Table 3, when the sample is 100 μL and the amount of dichloromethane is 300 μL, the amount of methanol used is in the range of 100 μL to 150 μL, i.e. the volume ratio of sample-methanol-dichloromethane is 1:1:3 to 1:1.5:3, and Oligo can obtain good extraction effect.

[0059] In summary, when using methanol-dichloromethane extractant, methanol is not easily dissolved in dichloromethane due to the large difference in polarity between methanol and dichloromethane, and can be more miscible with water in the sample, thereby obtaining a larger volume of supernatant. When the sample-methanol-dichloromethane ratio is in the range of 1:1.5:1 and 1:2:2~1:2.5:3, the concentration determination value of the supernatant obtained fluctuates greatly, and the supernatant will appear turbid after standing in the fume hood for 30 min.

[0060] Example 3 (1) Investigate the extraction effect of 100 μL dichloromethane and different amounts of ethanol extractant Take 100 μL of Oligo-LNP sample prepared in Example 1, a total of 18 portions, add 100 μL of dichloromethane, then add different amounts of ethanol (50 μL, 80 μL, 100 μL, 120 μL, 150 μL and 200 μL), each ethanol amount is prepared in triplicate, mix well, centrifuge at 12000 rpm for 2 min, take the supernatant, measure its volume, and use a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE) to measure the Oligo concentration, investigate the extraction effect of different amounts of ethanol and 100 μL dichloromethane extractant on Oligo, and the results are shown in Table 4.

[0061] Table 4 Extraction effect of different amounts of ethanol and 100 μL dichloromethane extractant on Oligo

[0062] As shown in Table 4, when the sample is 100 μL and the amount of dichloromethane is 100 μL, the amount of ethanol used is in the range of 50 μL~150 μL, i.e. the volume ratio of sample-ethanol-dichloromethane is 1:0.5:1~1:1.5:1, Oligo can obtain good extraction effect.

[0063] (2) Investigate the extraction effect of 200 μL dichloromethane and different amounts of ethanol extractant The Oligo-LNP sample prepared in Example 1 was measured at 100 μL, a total of 21 portions, and 200 μL of dichloromethane was added, and different amounts of ethanol (50 μL, 100 μL, 120 μL, 150 μL, 200 μL, 250 μL and 300 μL) were added, and each ethanol amount was prepared in triplicate, mixed, centrifuged at 12000 rpm for 2 min, the supernatant was taken, the volume was measured, and the Oligo concentration was measured using a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE), and the effect of different amounts of ethanol and 200 μL of dichloromethane on the extraction of Oligo was investigated, and the results are shown in Table 5.

[0064] Table 5 Effect of different amounts of ethanol and 200 μL of dichloromethane on the extraction of Oligo

[0065] As shown in Table 5, when the sample is 100 μL and the amount of dichloromethane is 200 μL, the amount of ethanol used is in the range of 50 μL to 200 μL, that is, the sample-ethanol-dichloromethane volume ratio is 1:0.5:2 to 1:2:2, and Oligo can obtain good extraction effect.

[0066] (3) Investigation of the extraction effect of 300 μL of dichloromethane and different amounts of ethanol The Oligo-LNP sample prepared in Example 1 was measured at 100 μL, a total of 21 portions, and 300 μL of dichloromethane was added, and different amounts of ethanol (50 μL, 100 μL, 120 μL, 150 μL, 200 μL, 250 μL and 300 μL) were added, and each ethanol amount was prepared in triplicate, mixed, centrifuged at 12000 rpm for 2 min, the supernatant was taken, the volume was measured, and the Oligo concentration was measured using a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE), and the effect of different amounts of ethanol and 300 μL of dichloromethane on the extraction of Oligo was investigated, and the results are shown in Table 6.

[0067] Table 6 Effect of different amounts of ethanol and 300 μL of dichloromethane on the extraction of Oligo

[0068] As shown in Table 6, when the sample is 100 μL and the amount of dichloromethane is 300 μL, the amount of ethanol used is in the range of 50 μL to 250 μL, that is, the sample-ethanol-dichloromethane volume ratio is 1:0.5:3 to 1:2.5:3, and Oligo can obtain good extraction effect.

[0069] In summary, the extraction results using ethanol and dichloromethane as the extractant show that the supernatant volume obtained by the ethanol-dichloromethane combination is smaller than that obtained by the methanol-dichloromethane combination, mainly because the polarity difference between ethanol and dichloromethane is smaller than that between methanol and dichloromethane, and ethanol can be more dissolved in dichloromethane. However, the ethanol-dichloromethane combination can obtain supernatant with higher Oligo concentration and smaller proportion of organic solvent.

[0070] Example 4 (1) Investigation of the extraction effect of the extractant composed of 100 μL dichloromethane and different amounts of isopropanol Take 100 μL of the Oligo-LNP sample prepared in Example 1, a total of 18 portions, add 100 μL of dichloromethane, and then add different amounts of isopropanol (50 μL, 80 μL, 100 μL, 150 μL, 180 μL and 200 μL), respectively. Each isopropanol amount is prepared in triplicate, mixed, centrifuged at 12000 rpm for 2 min, take the supernatant, measure its volume, and use a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE) to measure the Oligo concentration, to investigate the effect of the extractant composed of different amounts of isopropanol and 100 μL dichloromethane on the extraction of Oligo, and the results are shown in Table 7.

[0071] Table 7 Extraction effect of the extractant composed of different amounts of isopropanol and 100 μL dichloromethane on Oligo

[0072] As shown in Table 7, when the sample is 100 μL, the amount of isopropanol used is in the range of 50 μL to 150 μL, i.e. the sample-isopropanol-dichloromethane volume ratio is 1:0.5:1 to 1:1.5:1, Oligo can obtain good extraction effect.

[0073] (2) Investigation of the extraction effect of the extractant composed of 200 μL dichloromethane and different amounts of isopropanol Take 100 μL of the Oligo-LNP sample prepared in Example 1, a total of 18 portions, add dichloromethane 200 μL respectively, and then add different amounts of isopropyl alcohol (50 μL, 100 μL, 150 μL, 200 μL, 250 μL and 300 μL) respectively. Each isopropyl alcohol amount is prepared in triplicate. Mix well, centrifuge at 12000 rpm for 2 min, take the supernatant, measure its volume, and use a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE) to measure the Oligo concentration. The effect of different amounts of isopropyl alcohol and 200 μL of dichloromethane on the extraction of Oligo is investigated, and the results are shown in Table 8.

[0074] Table 8 Effect of different amounts of isopropyl alcohol and 200 μL of dichloromethane on the extraction of Oligo

[0075] As shown in Table 8, when the sample is 100 μL and the amount of dichloromethane is 200 μL, the amount of isopropyl alcohol used is in the range of 50 μL to 200 μL, i.e. the sample-isopropyl alcohol-dichloromethane volume ratio is 1:0.5:2 to 1:2:2, and Oligo can obtain good extraction effect.

[0076] (3) Investigation of the extraction effect of an extractant composed of 300 μL of dichloromethane and different amounts of isopropyl alcohol Take 100 μL of the Oligo-LNP sample prepared in Example 1, a total of 18 portions, add dichloromethane 300 μL respectively, and then add different amounts of isopropyl alcohol (50 μL, 100 μL, 150 μL, 200 μL, 250 μL and 300 μL) respectively. Each isopropyl alcohol amount is prepared in triplicate. Mix well, centrifuge at 12000 rpm for 2 min, take the supernatant, measure its volume, and use a micro UV spectrophotometer (Thermo Fisher, NANODROP ONE) to measure the Oligo concentration. The effect of different amounts of isopropyl alcohol and 300 μL of dichloromethane on the extraction of Oligo is investigated, and the results are shown in Table 9.

[0077] Table 9 Effect of different amounts of isopropyl alcohol and 300 μL of dichloromethane on the extraction of Oligo

[0078] As shown in Table 9, when the sample is 100 μL and the amount of dichloromethane is 300 μL, the amount of isopropyl alcohol used is in the range of 100 μL to 250 μL, i.e. the sample-isopropyl alcohol-dichloromethane volume ratio is 1:1:3 to 1:2.5:3, and Oligo can obtain good extraction effect.

[0079] Example 5 Effect of centrifugation time and centrifugation speed on Oligo extraction Take 100 μL of the Oligo-LNP sample prepared in Example 1, a total of 18 portions, add isopropyl alcohol 150 μL respectively, then add dichloromethane 200 μL respectively, set up: 10 min standing group, 12000 rpm centrifugation 1 min group, 12000 rpm centrifugation 2 min group, 12000 rpm centrifugation 5 min group, 4000 rpm centrifugation 2 min group, 8000 rpm centrifugation 2 min group, three replicates for each group, to investigate the effect of centrifugation speed and centrifugation time on extraction, the results are shown in Table 10.

[0080] Table 10 Effect of centrifugation time and centrifugation speed on Oligo extraction

[0081] As shown in Table 10, the centrifugation time and centrifugation speed have no effect on the extraction of Oligo.

[0082] Example 6 The difference between this embodiment and Example 1 is that the lipid component of the Oligo-LNP sample in this embodiment does not contain permanent cationic lipid DOTAP, the molar ratio of DODMA, DOPC, cholesterol, DMG-PEG-2K is 45:15:38:2.

[0083] The preparation method is the same as Example 1, and in the obtained Oligo-LNP, the total lipid concentration is about 16 mg / mL, the Oligo concentration is about 1.6 mg / mL, and the drug-lipid ratio is 1:10.

[0084] Example 7 Take 100 μL of the Oligo-LNP sample prepared in Example 6, add methanol 150 μL, then add dichloromethane 200 μL, mix well, centrifuge at 12000 rpm for 2 min, take the supernatant to determine the Oligo concentration. The results are shown in Table 11.

[0085] Table 11 Oligo extraction results of Oligo-LNP sample prepared in Example 6 with 150 μL methanol and 200 μL dichloromethane as extractant

[0086] Example 8 Take 100 μL of the Oligo-LNP sample prepared in Example 6, add 200 μL of dichloromethane, and then add 150 μL of ethanol, mix, centrifuge at 12000 rpm for 2 min, and then take the supernatant to determine the volume and Oligo concentration. The results are shown in Table 12.

[0087] Table 12 Oligo extraction results of the Oligo-LNP sample prepared in Example 6 using an extractant composed of 150 μL of ethanol and 200 μL of dichloromethane

[0088] Example 9 Take 100 μL of the Oligo-LNP sample prepared in Example 6, add 200 μL of dichloromethane, and then add 150 μL of isopropanol, mix, centrifuge at 12000 rpm for 2 min, and then take the supernatant to determine the volume and Oligo concentration. The results are shown in Table 13.

[0089] Table 13 Oligo extraction results of the Oligo-LNP sample prepared in Example 6 using an extractant composed of 150 μL of isopropanol and 200 μL of dichloromethane

[0090] Example 10 Unlike Example 1, the lipid component of the Oligo-LNP sample in this example is composed of SM-102, DOPC, cholesterol, and DMG-PEG-2K, and the molar ratio of SM-102, DOPC, cholesterol, and DMG-PEG-2K is 45:15:38:2.

[0091] The preparation method is the same as that in Example 1, and in the obtained Oligo-LNP, the total lipid concentration is about 16 mg / mL, the Oligo concentration is about 1.6 mg / mL, and the drug-lipid ratio is 1:10.

[0092] Example 11 Prepare an Oligo-LNP sample with a total lipid concentration of about 8 mg / mL and a drug-lipid ratio of 1:5, and the rest is the same as in Example 10.

[0093] Example 12 Prepare an Oligo-LNP sample with a total lipid concentration of about 32 mg / mL and a drug-lipid ratio of 1:20, and the rest is the same as in Example 10.

[0094] Example 13 Methanol and dichloromethane were used as reagents to extract Oligo from the Oligo-LNP samples prepared in Examples 10-12. Extraction method: 100 μL of sample was taken, 150 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 Oligo concentration. The extraction results are shown in Tables 14-16.

[0095] Table 14 Oligo extraction results of the Oligo-LNP sample prepared in Example 10

[0096] Table 15 Oligo extraction results of the Oligo-LNP sample prepared in Example 11

[0097] Table 16 Oligo extraction results of the Oligo-LNP sample prepared in Example 12

[0098] Experimental Example 1: Methodological Validation Based on the results of Examples 2-4, Oligo can achieve good extraction results in combinations of methanol-dichloromethane, ethanol-dichloromethane, and isopropanol-dichloromethane within a certain proportion range. The median reagent dosage of each combination was selected as the extraction method for methodology validation.

[0099] 1. Extraction of Oligo with Methanol-Dichloromethane Extraction method: Take 100 μL of the Oligo-LNP sample prepared in Example 1, add 200 μL of dichloromethane, then add 150 μL of methanol, mix well, centrifuge at 12000 rpm for 2 min, take the supernatant and determine the volume and Oligo concentration.

[0100] (1) Accuracy The theoretical concentration of Oligo in the Oligo-LNP sample is 1.6 mg / mL. Weigh out 59.80 mg (approximately equivalent to 48 mg net Oligo content), 39.93 mg (approximately equivalent to 32 mg net Oligo content), and 20.45 mg (approximately 16 mg net Oligo content) of Oligo reference standard (content 80.6%, ZJHC00A), respectively, and place them in 20 mL volumetric flasks. Dissolve and dilute to volume with blank LNP (containing no Oligo, other components are the same as in Example 1) to prepare 50%, 100%, and 150% accuracy solutions, respectively. Take three portions of each accuracy solution and extract Oligo using the extraction method described above. The extraction results are shown in Table 17.

[0101] Table 17 Methanol-dichloromethane extraction accuracy results

[0102] As shown in Table 17, the recovery rate of Oligo extracted by the method of the present application was 98.5%~100.8%, and the RSD of 9 accuracy solutions was 0.7%, indicating good accuracy.

[0103] (2) Reproducibility Six Oligo-LNP samples were measured and Oligo was extracted according to the above extraction method. The extraction results are shown in Table 18.

[0104] Table 18 Methanol-dichloromethane reproducibility results

[0105] As shown in Table 18, the extraction recovery rate of Oligo extracted by the method of the present application was 102.4%~104.5%, the average recovery rate was 103.4%, and the RSD was 0.7%, indicating good reproducibility.

[0106] (3) Intermediate precision Oligo-LNP samples were measured and Oligo was extracted according to the above extraction method. The extraction results are shown in Tables 19~20.

[0107] Table 19 Methanol-dichloromethane intermediate precision results

[0108] Table 20 Methanol-dichloromethane extraction results of 12 test samples

[0109] As shown in Table 19, the extraction recovery rate of Oligo extracted by the method of the present application was 101.8%~103.0%, the average recovery rate was 102.4%, and the RSD was 0.5%; as shown in Table 20, the extraction recovery rate of Oligo extracted by the method of the present application was 101.8%~104.5%, the average recovery rate was 102.9%, and the RSD was 0.8%, indicating good intermediate precision.

[0110] 2. Extraction of Oligo by ethanol-dichloromethane Extraction method: 100 μL of Oligo-LNP sample prepared in Example 1 was measured, 200 μL of dichloromethane was added, 150 μL of ethanol was added, and the mixture was mixed, centrifuged at 12000 rpm for 2 min, and the supernatant was measured for volume and Oligo concentration.

[0111] (1) Accuracy The accuracy solution of "1, methanol-dichloromethane extraction Oligo" was measured, three times each, and Oligo extraction was performed according to the above extraction method, and the extraction results are shown in Table 21.

[0112] Table 21 Ethanol-dichloromethane extraction accuracy results

[0113] As shown in Table 21, the recovery rate of Oligo extracted by the method of the present application was 96.1%~99.9%, and the RSD of 9 accuracy solutions was 1.3%, and the accuracy was good.

[0114] (2) Reproducibility The Oligo-LNP sample was measured 6 times, and Oligo extraction was performed according to the above extraction method, and the extraction results are shown in Table 22.

[0115] Table 22 Ethanol-dichloromethane reproducibility results

[0116] As shown in Table 22, the extraction recovery rate of Oligo by the method of the present application was 100.5%~105.6%, the average recovery rate was 102.5%, and the RSD was 1.9%, and the reproducibility was good.

[0117] (3) Intermediate precision The Oligo-LNP sample was measured, and Oligo extraction was performed according to the above extraction method, and the extraction results are shown in Tables 23~24.

[0118] Table 23 Ethanol-dichloromethane intermediate precision results

[0119] Table 24 Ethanol-dichloromethane 12 test sample extraction results

[0120] As shown in Table 23, the extraction recovery rate of Oligo by the method of the present application was 101.4%~104.3%, the average recovery rate was 103.0%, and the RSD was 1.0%; as shown in Table 24, the extraction recovery rate of Oligo by the method of the present application was 100.5%~105.6%, the average recovery rate was 102.7%, and the RSD was 1.5%, and the intermediate precision was good.

[0121] 3, isopropanol-dichloromethane extraction Oligo Extraction method: 100 μL of Oligo-LNP sample prepared in Example 1 was taken, 150 μL of isopropanol was added, 200 μL of dichloromethane was added, mixed, centrifuged at 12000 rpm for 2 min, and the supernatant was taken to determine the volume and Oligo concentration.

[0122] (1) Accuracy The accuracy solution in "1, methanol-dichloromethane extraction Oligo" was taken, three times each, and Oligo extraction was performed according to the above extraction method, and the extraction results are shown in Table 25.

[0123] Table 25 Isopropanol-dichloromethane extraction accuracy results

[0124] As shown in Table 25, the recovery rate of Oligo extracted by the method of the present application was 93.9%~101.7%, the RSD of 9 accuracy solutions was 2.3%, and the accuracy was good.

[0125] (2) Reproducibility Six Oligo-LNP samples were taken and Oligo extraction was performed according to the above extraction method, and the extraction results are shown in Table 26.

[0126] Table 26 Isopropanol-dichloromethane reproducibility results

[0127] As shown in Table 26, the extraction recovery rate of Oligo by the method of the present application was 102.2%~104.2%, the average recovery rate was 103.4%, and the RSD was 0.7%, and the reproducibility was good.

[0128] (3) Intermediate precision Oligo-LNP samples were taken and Oligo extraction was performed according to the above extraction method, and the extraction results are shown in Tables 27~28.

[0129] Table 27 Isopropanol-dichloromethane intermediate precision results

[0130] Table 28 Isopropanol-dichloromethane extraction results of 12 test samples

[0131] As shown in Table 27, the recovery rate of Oligo extracted by the method of the present application is 100.6%~103.9%, the average recovery rate is 102.2%, and the RSD is 1.1%; as shown in Table 28, the recovery rate of Oligo extracted by the method of the present application is 100.6%~104.2%, the average recovery rate is 102.8%, and the RSD is 1.1%, and the intermediate precision is good.

[0132] Test Example 2 Oligo-LNP sample related substance inspection 1. Chromatographic conditions including: Chromatographic column: ACQUITY UPLC ® BEH C18 1.7 µm, 2.1×100 mm, or equivalent chromatographic column; Mobile phase A: accurately measure 1000 mL of water, first add 2 mL of triethylamine, mix well, then add 10 mL of hexafluoroisopropanol, mix well, and ultrasonic degassing to obtain; Mobile phase B: accurately measure 750 mL of water, 250 mL of acetonitrile, first add 1 mL of triethylamine, mix well, then add 5 mL of hexafluoroisopropanol, mix well, and ultrasonic degassing to obtain; Detector: PDA or ultraviolet detector; detection wavelength: 260 nm; column temperature: 60°C; sample chamber temperature: room temperature; flow rate: 0.3 mL / min; injection volume: 15 μL; needle washing liquid: 10% methanol; Elution mode: gradient elution, the elution program is:

[0133] 2. Preparation of test sample Oligo drug substance (API, ZJHC00A) was weighed, dissolved and diluted with water to the injection concentration, and injected as an API control. API was dissolved in a blank LNP (without drug, other conditions consistent with Example 1), and Oligo was extracted according to the three pretreatment methods provided by the present application. The sample was injected as a pretreated sample, and the number and content of impurities were compared with the API control.

[0134] (1) Blank control: Example 1 blank LNP (without drug).

[0135] (2) API control solution: weigh 40 mg of Oligo drug substance (content of 80.6%) (about equivalent to 32 mg of net Oligo content), place in a 20 mL volumetric flask, dissolve and dilute with water, and then accurately take 1 mL into a 20 mL volumetric flask and dilute with water. As an API control solution without pretreatment method.

[0136] (3) Sample solution: 40 mg of Oligo API (content 80.6%) (about equivalent to 32 mg of Oligo net content) was weighed into a 20 mL volumetric flask, dissolved with blank LNP (without Oligo, other components consistent) and diluted to volume, as the sample solution.

[0137] (4) Sample pretreatment: the sample solution was pretreated by the following three methods: Method 1: 100 μL of sample solution was taken, 200 μL of dichloromethane was added, 150 μL of methanol was added, mixed, centrifuged at 12000 rpm for 2 min, 100 μL of supernatant was taken, 600 μL of water was added, 3 samples were prepared in parallel, and the related substances were determined according to the chromatographic conditions.

[0138] Method 2: 100 μL of sample solution was taken, 200 μL of dichloromethane was added, 150 μL of ethanol was added, mixed, centrifuged at 12000 rpm for 2 min, 100 μL of supernatant was taken, 700 μL of water was added, 3 samples were prepared in parallel, and the related substances were determined according to the chromatographic conditions.

[0139] Method 3: 100 μL of sample solution was taken, 150 μL of isopropyl alcohol was added, 200 μL of dichloromethane was added, mixed, centrifuged at 12000 rpm for 2 min, 100 μL of supernatant was taken, 1000 μL of water was added, 3 samples were prepared in parallel, and the related substances were determined according to the chromatographic conditions.

[0140] 3. Test results The chromatograms of the blank control and API control solution are shown in Figure 1 and 2 respectively.

[0141] (1) Impurity detection of samples pretreated by method 1 The related substance detection results of the sample solution pretreated by 200 μL of dichloromethane and 150 μL of methanol are shown in Table 29.

[0142] Table 29 Impurity detection results of samples pretreated by method 1

[0143] The chromatogram of test sample 1 in pretreatment method 1 is shown in Figure 3 .

[0144] As shown in Table 29, the RRT of 0.98 (peak No. 8) was the maximum single impurity, the maximum single impurity in the API control sample was 3.40%, the maximum single impurities of the three test samples treated by the pretreatment method 1 were 3.43%, 3.46% and 3.47% respectively, the recoveries were 100.9%-102.1%, and the recoveries were good. The purity of the API control sample was 92.47%, the purities of the three test samples treated by the pretreatment method 1 were 92.50%, 92.51% and 92.41% respectively, the recoveries were 99.9%-100.0%, and the recoveries were good. Moreover, all the impurities and the main peak were one-to-one corresponding, and there was no missing impurity.

[0145] (2) Impurity detection of the sample pretreated by the method 2 The impurity detection results of the sample solution pretreated by the extraction agent composed of 200 μL of dichloromethane and 150 μL of ethanol are shown in Table 30.

[0146] Table 30 Impurity detection results of the sample pretreated by the method 2

[0147] The chromatogram of the test sample 3 in the pretreatment method 2 is shown in Figure 4 .

[0148] As shown in Table 30, the RRT of 0.98 (peak No. 8) was the maximum single impurity, the maximum single impurity in the API control sample was 3.40%, the maximum single impurities of the three test samples treated by the pretreatment method 2 were 3.48%, 3.50% and 3.52% respectively, the recoveries were 102.4%-103.5%, and the recoveries were good. The purity of the API control sample was 92.47%, the purities of the three test samples treated by the pretreatment method 2 were 92.37%, 92.35% and 92.50% respectively, the recoveries were 99.9%-100.0%, and the recoveries were good. Moreover, all the impurities and the main peak were one-to-one corresponding, and there was no missing impurity.

[0149] (3) Impurity detection of the sample pretreated by the method 3 The impurity detection results of the sample solution pretreated by the extraction agent composed of 150 μL of isopropanol and 200 μL of dichloromethane are shown in Table 31.

[0150] Table 31 Impurity detection results of the sample pretreated by the method 3

[0151] The chromatogram of the test sample 2 in the pretreatment method 3 is shown in Figure 5 .

[0152] As shown in the results of Table 31, the RRT of 0.98 (peak No. 8) was the maximum single impurity, the maximum single impurity in the API control sample was 3.40%, the maximum single impurities of the three test samples treated by pretreatment method 3 were 3.52%, 3.52% and 3.52% respectively, and the recoveries were all 103.5%, which was good. The purity of the API control sample was 92.47%, the purities of the three test samples treated by pretreatment method 3 were 92.30%, 92.32% and 92.3% respectively, the recoveries were 99.8%-99.9%, which was good. And all the impurities and main peaks were one-to-one corresponding, and there was no missing impurity.

[0153] The above only describes some embodiments of the present application. For those skilled in the art, without departing from the inventive concept, several modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. A pre-treatment method for oligo-LNP samples, characterized in that, The method comprises the following steps: adding an extractant to the Oligo-LNP sample, standing or centrifuging, and taking the upper solution to obtain an Oligo solution; The extractant is composed of a first organic reagent and a second organic reagent; the first organic reagent is selected from alcohol organic solvents; and the second organic reagent is selected from halogenated hydrocarbon organic solvents.

2. The pretreatment method according to claim 1, wherein the alcohol organic solvent is selected from at least one of methanol, ethanol, isopropanol and butanol; and the halogenated hydrocarbon organic solvent is selected from dichloromethane, trichloromethane or a combination thereof.

3. The pre-treatment method according to claim 2, characterized in that, The concentration of total lipids in the Oligo-LNP sample is 1.6-32 mg / mL.

4. The pre-treatment method according to claim 3, characterized in that, The mass ratio of Oligo to total lipids in the Oligo-LNP sample is 1:5-1:

40.

5. The pre-treatment method according to claim 4, characterized in that, The volume ratio of the Oligo-LNP sample, the first organic reagent and the second organic reagent is 1:(0.5-2.5):(1-3).

6. The pre-treatment method according to any one of claims 1 to 5, characterized in that, The lipid component of the Oligo-LNP sample comprises ionizable cationic lipids, phosphogroup-containing neutral lipids, sterol lipids and polymer-conjugated lipids.

7. The pre-treatment method according to claim 6, characterized in that, The ionizable cationic lipids are selected from one or more of DODMA, DLin-MC3-DMA, ALC-0315, SM-102, YK-201, PL40, VN2C1, C12-200, Lipid HTO12, TNT-b10, Lipid 20b, C14-4, 5A2-SC8, CKK-E12, DLin-KC2-DMA, 306Oi10, TCL053, 306-O12B, GenevantCL1, L319, OF-C4-Deg-Lin, PPZ-A10, Ionizable lipid-1, OF-02, Lipid 331, OF-Deg-Lin, ATX-002, 244cis, 113-N16B, Ionizable lipid-2, AA3-Dlin, C10-200, 113-O12B, A12-Iso5-2DC18, 4A3-Cit, RCB-02-4-8, Lipid AX4, Lipid DIM1, 9322-O16B, Al-28, 306Oi9-cis2.

8. The pre-treatment method according to claim 6, characterized in that, The phosphogroup-containing neutral lipids are selected from one or more of distearoylphosphatidylcholine, egg yolk lecithin, soybean phospholipid, hydrogenated soybean phospholipid, dioleoylphosphatidylethanolamine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoylphosphatidylcholine, diarachidoylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoyl ethanolamine, 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol and 1-hexadecan 2-(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine.

9. The pre-treatment method according to claim 6, characterized in that, The solid sterol lipid is selected from one or more of β-sitosterol, stigmasterol, campesterol, cholesterol, and sterol derivatives.

10. The pre-treatment method according to claim 6, characterized in that, The polymer-conjugated lipid is selected from one or more of a polyethylene glycol-conjugated lipid.

11. The pre-treatment method according to claim 10, characterized in that, The polyethylene glycol-conjugated lipid is selected from one or more of PEG-dodecyl acetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-phospholipid, PEG-ceramide, DMG-PEG, DSPE-PEG, and DSG-PEG.

12. The pretreatment method according to any one of claims 7 to 11, characterized in that, The lipid component of the Oligo-LNP sample further comprises a permanent cationic lipid.

13. The pre-treatment method according to claim 12, 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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