Method of analysing lipid nanoparticles in physiological fluids

Abstract

Methods and systems for analyzing lipid nanoparticles using size exclusion chromatography in combination with multi-angle light scattering are disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 379,852, filed on October 17, 2022. The contents of the above application are hereby incorporated by reference in their entirety. Background Art

[0003] As one of the breakthrough technologies after the COVID-19 pandemic, lipid nanoparticles (mRNA-LNP) loaded with mRNA have been proven to be a safe and effective way to express proteins in human cells and tissues without mutagenic risks. LNP has also been used for clinical delivery of siRNA and is currently studying many clinical applications beyond vaccination. This includes cancer immunotherapy and rare genetic diseases. In order to expand the production capacity of approved lipid excipients and in order to discover new LNP formulations with improved properties, a large amount of resources have been allocated. Therefore, there is a need for developing new methods and systems for quantitatively analyzing lipid nanoparticles in the presence of physiological fluids. Summary of the invention

[0004] In one aspect, the present disclosure provides a method for analyzing lipid nanoparticles (LNPs). The method comprises: (optionally) obtaining a sample comprising LNPs (e.g., in a physiological fluid); performing size exclusion chromatography (SEC) on the sample; and obtaining a multi-angle light scattering (MALS) signal from the sample to analyze the LNPs.

[0005] In some embodiments, the method further comprises: performing SEC on a reference sample; and obtaining a MALS signal from the reference sample. In some embodiments, the method further comprises comparing the MALS signal from the sample with the MALS signal from the reference sample.

[0006] In some embodiments, the method further comprises performing LC-MS / MS to the sample, e.g., determining (e.g., quantitatively) the components of the LNP. In some embodiments, the method further comprises obtaining the UV absorbance of the sample (e.g., at λ=260nm), e.g., determining (e.g., quantitatively) the amount of nucleic acid in the LNP.

[0007] In some embodiments, the method determines the stability of the LNP in the sample. In some embodiments, the method determines the purity of the LNP in the sample. In some embodiments, the method is applicable to monitoring the preparation of the LNP. In some embodiments, the method is applicable to determining the pharmacokinetics (PK) of the LNP.

[0008] In some embodiments, the sample is not obtained from a subject, e.g., a physiological fluid reconstituted with LNPs. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject is a healthy subject. In some embodiments, the subject suffers from or may suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder. In some embodiments, the subject is a human. In some embodiments, the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0009] In some embodiments, SEC is carried out in a single column configuration. In some embodiments, SEC is carried out in a dual column configuration, for example, using two columns with different pore sizes, for example, a first column with a first average pore size and a second column with a second average pore size. In some embodiments, the first average pore size is greater than the second average pore size. In some embodiments, the first average pore size has a molecular weight cutoff (MWCO) of about 1 MDa or more, for example, about 2 MDa or more, 5 MDa or more, 10 MDa or more, 15 MDa or more or 20 MDa or more. In some embodiments, the first average pore size has a MWCO of about 20 MDa or less, for example, about 15 MDa or less, 10 MDa or less, 5 MDa or less, 2 MDa or less or 1 MDa or less. In some embodiments, the first average pore size has a MWCO of about 1 MDa to 20 MDa, for example, about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, for example, about 10 MDa. In some embodiments, the second average pore size has a MWCO of about 10 MDa or less, for example, about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less, or 0.1 MDa or less. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa or greater, e.g., about 0.2 MDa or greater, 0.5 MDa or greater, 1 MDa or greater, 2 MDa or greater, 5 MDa or greater, or 10 MDa or greater. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa to 10 MDa, for example, about 0.2 MDa to 5 MDa, 0.5 MDa to 2 MDa, 0.1 MDa to 5 MDa, 0.1 MDa to 2 MDa, 0.1 MDa to 1 MDa, 0.1 MDa to 0.5 MDa, 0.1 MDa to 0.2 MDa, 5 MDa to 10 MDa, 2 MDa to 10 MDa, 1 MDa to 10 MDa, 0.5 MDa to 10 MDa, 0.2 MDa to 10 MDa, 0.2 MDa to 1 MDa, 1 MDa to 5 MDa, or 0.3 MDa to 0.7 MDa, for example, about 0.5 MDa.

[0010] In some embodiments, SEC is performed using a column having a length of 100 mm or more, e.g., about 150 mm or more, 200 mm or more, 250 mm or more, 300 mm or more, 400 mm or more, 500 mm or more, 600 mm or more, 700 mm or more, 800 mm or more, 900 mm or more, or 1000 mm or more, e.g., about 100 mm to 1000 mm, 150 mm to 500 mm, or about 200 mm to 300 mm, e.g., about 250 mm.

[0011] In some embodiments, SEC is performed using a polymer-based column. In some embodiments, SEC is not performed using a silica-based column.

[0012] In some embodiments, SEC is performed under aqueous conditions, e.g., using a biocompatible buffer system, e.g., phosphate buffer, bicarbonate buffer, acetate buffer, Tris buffer, or Good's buffer (e.g., as described in Good et al. Biochemistry. 1966; 5(2):467-77; Good and Izawa Methods Enzymol. 1972; 24:53-68; Ferguson et al. Anal. Biochem. 1980 May; 104 (2): 300-10) any one, for example, MES, Bis-tris methane, ADA, Bis-tris propane, PIPES, ACES, MOPSO, choline chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetylaminoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tris (hydroxymethyl) methylglycine (Tricine), tris hydroxymethylaminomethane (Tris), glycine amide, glycine peptide, HEPBS, N-bihydroxyethylglycine (Bicine), TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS or any one of a combination thereof. In some embodiments, SEC is performed without using an organic solvent (e.g., THF) as the mobile phase.

[0013] In some embodiments, MALS is performed at scattering angles of 15° and 90°. In some embodiments, MALS is performed using a laser wavelength of about 500nm to 800nm, e.g., about 600nm to 700nm, 500nm to 700nm, 600nm to 800nm, 500nm to 600nm, 700nm to 800nm, e.g., about 658nm. In some embodiments, MALS is performed using a sample cell volume of about 1 μL to 50 μL, e.g., about 2 μL to 25 μL, 5 μL to 20 μL, 1 μL to 40 μL, 1 μL to 30 μL, 1 μL to 20 μL, 1 μL to 10 μL, 40 μL to 50 μL, 30 μL to 50 μL, 20 μL to 50 μL, 10 μL to 50 μL, 5 μL to 15 μL, or 10 μL to 20 μL, e.g., about 10 μL. In some embodiments, MALS is performed using a scattering volume of about 0.001 μL to 0.1 μL, e.g., about 0.005 μL to 0.05 μL, 0.001 μL to 0.05 μL, 0.001 μL to 0.01 μL, 0.05 μL to 0.1 μL, 0.01 μL to 0.1 μL, 0.005 μL to 0.1 μL, or 0.005 μL to 0.05 μL, e.g., about 0.01 μL. In some embodiments, MALS is performed at a temperature range of about 20°C to 70°C, e.g., about 25°C to 75°C or 30°C to 60°C. In some embodiments, MALS is performed with a temperature stability of no more than ±1°C, e.g., no more than ±0.5°C or ±0.2°C. In some embodiments, MALS is performed at a pH range of about 1-12, e.g., about 2-11 or 2-10.

[0014] In some embodiments, the size of the LNP is determined, for example, the hydrodynamic radius (R h In some embodiments, the molecular weight (MW) of the LNP is determined, for example, the weight average MW (MW) is determined. w ), number average MW(M n ), MW corresponding to the maximum value of the chromatographic peak (M p ), z-mean MW(M z or M z+1 ) or viscosity average MW (M v ) in one or more (e.g., 2, 3, 4, or 5). In some embodiments, the polydispersity index is determined, for example, by calculating M w With M n In some embodiments, determining the R of LNP gw, for example, using Zimm or partial Zimm methods (e.g., as described in Wyatt.AnalyticaChimicaActa.1993.272:1-40). In some embodiments, the half-life of the LNP is determined. In some embodiments, the stability of the LNP is determined according to the methods described herein, for example, the methods described in Example 1.

[0015] In some embodiments, the method comprises directly comparing the obtained MALS signals. In some embodiments, the method comprises indirectly comparing the obtained MALS signals.

[0016] In some embodiments, a plurality of LNPs in a sample are analyzed, eg, according to the methods described herein.

[0017] In some embodiments, the method does not include the step of recovering LNP, for example, by ultracentrifugation. In some embodiments, the method does not include the step of diluting the sample, for example, removing the high molecular weight components (for example, plasma or serum components) that interfere with detection (for example, by DLS detection). In some embodiments, the method does not include the step of labeling LNP, for example, the step of labeling LNP with a fluorophore. In some embodiments, the method quantitatively detects the LNP in the sample.

[0018] In some embodiments, LNP loads nucleic acid. In some embodiments, LNP does not load nucleic acid. In some embodiments, the nucleic acid is a therapeutic nucleic acid (TNA). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a vaccine. In some embodiments, the nucleic acid is a non-coding RNA, for example, a small non-coding RNA. In some embodiments, the nucleic acid is a small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or a micro RNA (miRNA). In some embodiments, the nucleic acid is a guide RNA (gRNA). In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid includes one or more modified nucleotides. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded.

[0019] In some embodiments, the sample comprises a physiological fluid. In some embodiments, the physiological fluid is plasma. In some embodiments, the physiological fluid is serum. In some embodiments, the physiological fluid is blood. In some embodiments, the physiological fluid is amniotic fluid, aqueous humor, bile, breast milk, cerebrospinal fluid, cerumen, chyle, exudate, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural effusion, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine or vomit. In some embodiments, the physiological fluid has a pH of 2-10, for example, 3-9, 4-8, 5-7, 2-8, 2-6, 2-4, 8-10, 6-10, 4-10, 2-10, 2-4, 3-5, 4-6, 6-8 or 7-9 pH. In some embodiments, the physiological fluid has the pH of serum or plasma (for example, 7.3-7.5). In some embodiments, the physiological fluid has the pH of a tumor microenvironment (eg, pH 5.6 to 6.8).

[0020] In another aspect, the present disclosure provides a method for determining the stability of lipid nanoparticles (LNPs) in a subject. The method comprises: (optionally) obtaining a first sample from a subject; performing size exclusion chromatography (SEC) on the first sample; obtaining a multi-angle light scattering (MALS) signal from the first sample; (optionally) obtaining a second sample comprising LNPs from a subject; performing SEC on the second sample; obtaining a second MALS signal from the second sample; and comparing the first MALS signal with the second MALS signal, wherein the comparison between the first MALS signal and the second MALS signal indicates the stability of the LNPs in the subject, thereby determining the stability of the LNPs in the subject.

[0021] In some embodiments, the subject has been administered LNPs, e.g., at least 1, 6, 12, 18, or 24 hours or 1, 2, 3, 4, 5, 6, or 7 days prior to obtaining the first sample comprising LNPs. In some embodiments, the second sample is obtained after obtaining the first sample, e.g., at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after obtaining the first sample.

[0022] In some embodiments, the method further comprises: (optionally) obtaining a third sample comprising LNP from the subject; performing SEC on the third sample; obtaining a third MALS signal from the third sample; and comparing the third MALS signal to the first MALS signal, the second MALS signal, or both, wherein the comparison indicates the stability of the LNP in the subject. In some embodiments, the third sample is obtained after the second sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the second sample is obtained.

[0023] In some embodiments, the method further comprises: (optionally) obtaining a fourth sample comprising LNP from a subject; performing SEC on the fourth sample; obtaining a fourth MALS signal from the fourth sample; and comparing the fourth MALS signal with one or more (e.g., all) of the first MALS signal, the second MALS signal, or the third MALS signal, wherein the comparison indicates the stability of the LNP in the subject. In some embodiments, the fourth sample is obtained after the third sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the third sample is obtained.

[0024] In some embodiments, the method further comprises: (optionally) obtaining a fifth sample comprising LNP from the subject; performing SEC on the fifth sample; obtaining a fifth MALS signal from the fifth sample; and comparing the fifth MALS signal with one or more (e.g., all) of the first MALS signal, the second MALS signal, the third MALS signal, or the fourth MALS signal, wherein the comparison indicates the stability of the LNP in the subject. In some embodiments, the fifth sample is obtained after obtaining the fourth sample, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after obtaining the fourth sample.

[0025] In some embodiments, the LNPs in the second or subsequent sample have a change in LNP composition, e.g., lipid, nucleic acid, or both, compared to the LNPs in the first or previous sample, optionally wherein the size (e.g., R h) and the size of the LNPs in the first or previous sample (e.g., R h ) are basically the same.

[0026] In some embodiments, the subject is a healthy subject. In some embodiments, the subject suffers from or may suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder. In some embodiments, the subject is a human. In some embodiments, the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0027] In some embodiments, SEC is carried out in a single column configuration. In some embodiments, SEC is carried out in a dual column configuration, for example, using two columns with different pore sizes, for example, a first column with a first average pore size and a second column with a second average pore size. In some embodiments, the first average pore size is greater than the second average pore size. In some embodiments, the first average pore size has a molecular weight cutoff (MWCO) of about 1 MDa or more, for example, about 2 MDa or more, 5 MDa or more, 10 MDa or more, 15 MDa or more or 20 MDa or more. In some embodiments, the first average pore size has a MWCO of about 20 MDa or less, for example, about 15 MDa or less, 10 MDa or less, 5 MDa or less, 2 MDa or less or 1 MDa or less. In some embodiments, the first average pore size has a MWCO of about 1 MDa to 20 MDa, for example, about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, for example, about 10 MDa. In some embodiments, the second average pore size has a MWCO of about 10 MDa or less, for example, about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less, or 0.1 MDa or less. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa or greater, e.g., about 0.2 MDa or greater, 0.5 MDa or greater, 1 MDa or greater, 2 MDa or greater, 5 MDa or greater, or 10 MDa or greater. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa to 10 MDa, for example, about 0.2 MDa to 5 MDa, 0.5 MDa to 2 MDa, 0.1 MDa to 5 MDa, 0.1 MDa to 2 MDa, 0.1 MDa to 1 MDa, 0.1 MDa to 0.5 MDa, 0.1 MDa to 0.2 MDa, 5 MDa to 10 MDa, 2 MDa to 10 MDa, 1 MDa to 10 MDa, 0.5 MDa to 10 MDa, 0.2 MDa to 10 MDa, 0.2 MDa to 1 MDa, 1 MDa to 5 MDa, or 0.3 MDa to 0.7 MDa, for example, about 0.5 MDa.

[0028] In some embodiments, SEC is performed using a column having a length of 100 mm or more, e.g., about 150 mm or more, 200 mm or more, 250 mm or more, 300 mm or more, 400 mm or more, 500 mm or more, 600 mm or more, 700 mm or more, 800 mm or more, 900 mm or more, or 1000 mm or more, e.g., about 100 mm to 1000 mm, 150 mm to 500 mm, or about 200 mm to 300 mm, e.g., about 250 mm.

[0029] In some embodiments, SEC is performed using a polymer matrix column. In some embodiments, SEC is not performed using a silica matrix column.

[0030] In some embodiments, SEC is performed under aqueous conditions, e.g., using a biocompatible buffer system, e.g., phosphate buffer, bicarbonate buffer, acetate buffer, Tris buffer, or Good's buffer (e.g., as described in Good et al. Biochemistry. 1966; 5(2):467-77; Good and Izawa Methods Enzymol. 1972; 24:53-68; Ferguson et al. Anal. Biochem. 1980 May; 104 (2): 300-10) any one, for example, MES, Bis-tris methane, ADA, Bis-tris propane, PIPES, ACES, MOPSO, choline chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetylaminoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tris (hydroxymethyl) methylglycine (Tricine), tris hydroxymethylaminomethane (Tris), glycine amide, glycine peptide, HEPBS, N-bihydroxyethylglycine (Bicine), TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS or any one of a combination thereof. In some embodiments, SEC is performed without using an organic solvent (e.g., THF) as the mobile phase.

[0031] In some embodiments, MALS is performed at scattering angles of 15° and 90°. In some embodiments, MALS is performed using a laser wavelength of about 500nm to 800nm, e.g., about 600nm to 700nm, 500nm to 700nm, 600nm to 800nm, 500nm to 600nm, 700nm to 800nm, e.g., about 658nm. In some embodiments, MALS is performed using a sample cell volume of about 1 μL to 50 μL, e.g., about 2 μL to 25 μL, 5 μL to 20 μL, 1 μL to 40 μL, 1 μL to 30 μL, 1 μL to 20 μL, 1 μL to 10 μL, 40 μL to 50 μL, 30 μL to 50 μL, 20 μL to 50 μL, 10 μL to 50 μL, 5 μL to 15 μL, or 10 μL to 20 μL, e.g., about 10 μL. In some embodiments, MALS is performed using a scattering volume of about 0.001 μL to 0.1 μL, e.g., about 0.005 μL to 0.05 μL, 0.001 μL to 0.05 μL, 0.001 μL to 0.01 μL, 0.05 μL to 0.1 μL, 0.01 μL to 0.1 μL, 0.005 μL to 0.1 μL, or 0.005 μL to 0.05 μL, e.g., about 0.01 μL. In some embodiments, MALS is performed at a temperature range of about 20°C to 70°C, e.g., about 25°C to 75°C or 30°C to 60°C. In some embodiments, MALS is performed with a temperature stability of no more than ±1°C, e.g., no more than ±0.5°C or ±0.2°C. In some embodiments, MALS is performed at a pH range of about 1-12, e.g., about 2-11 or 2-10.

[0032] In some embodiments, the size of the LNP is determined, for example, the hydrodynamic radius (R h In some embodiments, the molecular weight (MW) of the LNP is determined, for example, the weight average MW (MW) is determined. w ), number average MW(M n ), MW corresponding to the maximum value of the chromatographic peak (M p ), z-mean MW(M z or M z+1 ) or viscosity average MW (M v ) in one or more (e.g., 2, 3, 4, or 5). In some embodiments, the polydispersity index is determined, for example, by calculating M w With M n In some embodiments, determining the R of LNP gw, for example, using Zimm or partial Zimm methods (e.g., as described in Wyatt.AnalyticaChimicaActa.1993.272:1-40). In some embodiments, the half-life of the LNP is determined. In some embodiments, the stability of the LNP is determined according to the methods described herein, for example, the methods described in Example 1.

[0033] In some embodiments, the method comprises directly comparing the obtained MALS signals. In some embodiments, the method comprises indirectly comparing the obtained MALS signals.

[0034] In some embodiments, a plurality of LNPs in the sample are analyzed, eg, according to the methods described herein.

[0035] In some embodiments, the method does not include the step of recovering LNP, for example, by ultracentrifugation. In some embodiments, the method does not include the step of diluting the sample, for example, removing the high molecular weight components (for example, plasma or serum components) that interfere with detection (for example, by DLS detection). In some embodiments, the method does not include the step of labeling LNP, for example, the step of labeling LNP with a fluorophore. In some embodiments, the method quantitatively detects the LNP in the sample.

[0036] In some embodiments, LNP loads nucleic acid. In some embodiments, LNP does not load nucleic acid. In some embodiments, the nucleic acid is a therapeutic nucleic acid (TNA). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a vaccine. In some embodiments, the nucleic acid is a non-coding RNA, for example, a small non-coding RNA. In some embodiments, the nucleic acid is a small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or a micro RNA (miRNA). In some embodiments, the nucleic acid is a guide RNA (gRNA). In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid includes one or more modified nucleotides. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded.

[0037] In some embodiments, the sample comprises a physiological fluid. In some embodiments, the physiological fluid is plasma. In some embodiments, the physiological fluid is serum. In some embodiments, the physiological fluid is blood. In some embodiments, the physiological fluid is amniotic fluid, aqueous humor, bile, breast milk, cerebrospinal fluid, cerumen, chyle, exudate, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural effusion, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine or vomit. In some embodiments, the physiological fluid has a pH of 2-10, for example, 3-9, 4-8, 5-7, 2-8, 2-6, 2-4, 8-10, 6-10, 4-10, 2-10, 2-4, 3-5, 4-6, 6-8 or 7-9 pH. In some embodiments, the physiological fluid has the pH of serum or plasma (for example, 7.3-7.5). In some embodiments, the physiological fluid has the pH of a tumor microenvironment (eg, pH 5.6 to 6.8).

[0038] In another aspect, the present disclosure provides a method for determining the stability of lipid nanoparticles (LNPs) in a sample. The method comprises: (optionally) obtaining a first aliquot of the sample; performing size exclusion chromatography (SEC) on the first aliquot; obtaining a multi-angle light scattering (MALS) signal from the first aliquot; (optionally) obtaining a second aliquot of the sample; performing SEC on the second aliquot; obtaining a second MALS signal from the second aliquot; and comparing the first MALS signal with the second MALS signal, wherein the comparison between the first MALS signal and the second MALS signal indicates the stability of the LNPs in the sample, thereby determining the stability of the LNPs in the sample.

[0039] In some embodiments, the second aliquot is obtained after the first aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the first aliquot is obtained.

[0040] In some embodiments, the method further comprises: (optionally) obtaining a third aliquot of the sample; performing SEC on the third aliquot; obtaining a third MALS signal from the third aliquot; and comparing the third MALS signal to the first MALS signal, the second MALS signal, or both, wherein the comparison indicates the stability of the LNP in the sample. In some embodiments, the third aliquot is obtained after the second aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the second aliquot is obtained.

[0041] In some embodiments, the method further comprises: (optionally) obtaining a fourth aliquot of the sample; performing SEC on the fourth aliquot; obtaining a fourth MALS signal from the fourth aliquot; and comparing the fourth MALS signal with one or more (e.g., all) of the first MALS signal, the second MALS signal, or the third MALS signal, wherein the comparison indicates the stability of the LNP in the sample. In some embodiments, the fourth aliquot is obtained after obtaining the third aliquot, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after obtaining the third aliquot.

[0042] In some embodiments, the method further comprises: (optionally) obtaining a fifth aliquot of the sample; performing SEC on the fifth aliquot; obtaining a fifth MALS signal from the fifth aliquot, and comparing the fifth MALS signal with one or more (e.g., all) of the first MALS signal, the second MALS signal, the third MALS signal, or the fourth MALS signal, wherein the comparison indicates the stability of the LNP in the sample. In some embodiments, the fifth aliquot is obtained after obtaining the fourth aliquot, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after obtaining the fourth aliquot.

[0043] In some embodiments, a second or subsequent aliquot has a change in LNP composition, e.g., lipid, nucleic acid, or both, compared to the LNPs in the first or previous aliquot, optionally wherein the size (e.g., R h ) is related to the size of the LNPs in the first or previous aliquot (e.g., R h ) are basically the same.

[0044] In some embodiments, the sample is not obtained from a subject, e.g., a physiological fluid reconstituted with LNPs. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject is a healthy subject. In some embodiments, the subject suffers from or may suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder. In some embodiments, the subject is a human. In some embodiments, the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0045] In another aspect, the present disclosure provides a method for determining the purity of lipid nanoparticles (LNPs) in a sample. The method comprises: (optionally) obtaining a first aliquot of the sample; performing size exclusion chromatography (SEC) on the first aliquot; obtaining a multi-angle light scattering (MALS) signal from the first aliquot; (optionally) obtaining a second aliquot of the sample; performing SEC on the second aliquot; obtaining a second MALS signal from the second aliquot; and comparing the first MALS signal with the second MALS signal to determine the stability of the LNP in the sample, which indicates the purity of the LNP in the sample, thereby determining the purity of the LNP in the sample.

[0046] In some embodiments, the second aliquot is obtained after the first aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the first aliquot is obtained.

[0047] In some embodiments, the method further includes: (optionally) obtaining a first reference aliquot of a reference sample comprising LNPs; performing SEC on the first aliquot; obtaining a MALS signal from the first reference aliquot; (optionally) obtaining a second reference aliquot of the reference sample; performing SEC on the second reference aliquot; obtaining a second MALS signal from the second reference aliquot; and comparing the first MALS signal to the second MALS signal to determine the stability of the LNPs in the reference sample.

[0048] In some embodiments, the method further comprises comparing the stability of the LNP in the sample with the stability of the LNP in a reference sample, thereby determining the purity of the LNP in the sample.

[0049] In some embodiments, the method further comprises: (optionally) obtaining a third aliquot of the sample (or reference sample); performing SEC on the third aliquot; obtaining a third MALS signal from the third aliquot; and comparing the third MALS signal to the first MALS signal, the second MALS signal, or both. In some embodiments, the third aliquot is obtained after the second aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the second aliquot is obtained.

[0050] In some embodiments, the method further comprises: (optionally) obtaining a fourth aliquot of the sample (or reference sample); performing SEC on the fourth aliquot; obtaining a fourth MALS signal from the fourth aliquot; and comparing the fourth MALS signal to one or more (e.g., all) of the first MALS signal, the second MALS signal, or the third MALS signal. In some embodiments, the fourth aliquot is obtained after the third aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the third aliquot is obtained.

[0051] In some embodiments, the method further comprises: (optionally) obtaining a fifth aliquot of the sample (or reference sample); performing SEC on the fifth aliquot; obtaining a fifth MALS signal from the fifth aliquot; and comparing the fifth MALS signal to one or more (e.g., all) of the first MALS signal, the second MALS signal, the third MALS signal, or the fourth MALS signal. In some embodiments, the fifth aliquot is obtained after obtaining the fourth aliquot, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after obtaining the fourth aliquot.

[0052] In some embodiments, the LNPs in the sample have impurities due to incomplete deprotection of the LNPs.

[0053] In some embodiments, the sample is not obtained from a subject, e.g., a physiological fluid reconstituted with LNPs. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject is a healthy subject. In some embodiments, the subject suffers from or may suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder. In some embodiments, the subject is a human. In some embodiments, the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0054] In some embodiments, SEC is carried out in a single column configuration. In some embodiments, SEC is carried out in a dual column configuration, for example, using two columns with different pore sizes, for example, a first column with a first average pore size and a second column with a second average pore size. In some embodiments, the first average pore size is greater than the second average pore size. In some embodiments, the first average pore size has a molecular weight cutoff (MWCO) of about 1 MDa or more, for example, about 2 MDa or more, 5 MDa or more, 10 MDa or more, 15 MDa or more or 20 MDa or more. In some embodiments, the first average pore size has a MWCO of about 20 MDa or less, for example, about 15 MDa or less, 10 MDa or less, 5 MDa or less, 2 MDa or less or 1 MDa or less. In some embodiments, the first average pore size has a MWCO of about 1 MDa to 20 MDa, for example, about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, for example, about 10 MDa. In some embodiments, the second average pore size has a MWCO of about 10 MDa or less, for example, about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less, or 0.1 MDa or less. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa or greater, e.g., about 0.2 MDa or greater, 0.5 MDa or greater, 1 MDa or greater, 2 MDa or greater, 5 MDa or greater, or 10 MDa or greater. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa to 10 MDa, for example, about 0.2 MDa to 5 MDa, 0.5 MDa to 2 MDa, 0.1 MDa to 5 MDa, 0.1 MDa to 2 MDa, 0.1 MDa to 1 MDa, 0.1 MDa to 0.5 MDa, 0.1 MDa to 0.2 MDa, 5 MDa to 10 MDa, 2 MDa to 10 MDa, 1 MDa to 10 MDa, 0.5 MDa to 10 MDa, 0.2 MDa to 10 MDa, 0.2 MDa to 1 MDa, 1 MDa to 5 MDa, or 0.3 MDa to 0.7 MDa, for example, about 0.5 MDa.

[0055] In some embodiments, SEC is performed using a column having a length of 100 mm or more, e.g., about 150 mm or more, 200 mm or more, 250 mm or more, 300 mm or more, 400 mm or more, 500 mm or more, 600 mm or more, 700 mm or more, 800 mm or more, 900 mm or more, or 1000 mm or more, e.g., about 100 mm to 1000 mm, 150 mm to 500 mm, or about 200 mm to 300 mm, e.g., about 250 mm.

[0056] In some embodiments, SEC is performed using a polymer matrix column. In some embodiments, SEC is not performed using a silica matrix column.

[0057] In some embodiments, SEC is performed under aqueous conditions, e.g., using a biocompatible buffer system, e.g., phosphate buffer, bicarbonate buffer, acetate buffer, Tris buffer, or Good's buffer (e.g., as described in Good et al. Biochemistry. 1966; 5(2):467-77; Good and Izawa Methods Enzymol. 1972; 24:53-68; Ferguson et al. Anal. Biochem. 1980 May; 104 (2): 300-10) any one, for example, MES, Bis-tris methane, ADA, Bis-tris propane, PIPES, ACES, MOPSO, choline chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetylaminoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tris (hydroxymethyl) methylglycine (Tricine), tris hydroxymethylaminomethane (Tris), glycine amide, glycine peptide, HEPBS, N-bihydroxyethylglycine (Bicine), TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS or any one of a combination thereof. In some embodiments, SEC is performed without using an organic solvent (e.g., THF) as the mobile phase.

[0058] In some embodiments, MALS is performed at scattering angles of 15° and 90°. In some embodiments, MALS is performed using a laser wavelength of about 500nm to 800nm, e.g., about 600nm to 700nm, 500nm to 700nm, 600nm to 800nm, 500nm to 600nm, 700nm to 800nm, e.g., about 658nm. In some embodiments, MALS is performed using a sample cell volume of about 1 μL to 50 μL, e.g., about 2 μL to 25 μL, 5 μL to 20 μL, 1 μL to 40 μL, 1 μL to 30 μL, 1 μL to 20 μL, 1 μL to 10 μL, 40 μL to 50 μL, 30 μL to 50 μL, 20 μL to 50 μL, 10 μL to 50 μL, 5 μL to 15 μL, or 10 μL to 20 μL, e.g., about 10 μL. In some embodiments, MALS is performed using a scattering volume of about 0.001 μL to 0.1 μL, e.g., about 0.005 μL to 0.05 μL, 0.001 μL to 0.05 μL, 0.001 μL to 0.01 μL, 0.05 μL to 0.1 μL, 0.01 μL to 0.1 μL, 0.005 μL to 0.1 μL, or 0.005 μL to 0.05 μL, e.g., about 0.01 μL. In some embodiments, MALS is performed at a temperature range of about 20°C to 70°C, e.g., about 25°C to 75°C or 30°C to 60°C. In some embodiments, MALS is performed with a temperature stability of no more than ±1°C, e.g., no more than ±0.5°C or ±0.2°C. In some embodiments, MALS is performed at a pH range of about 1-12, e.g., about 2-11 or 2-10.

[0059] In some embodiments, the size of the LNP is determined, for example, the hydrodynamic radius (R h In some embodiments, the molecular weight (MW) of the LNP is determined, for example, the weight average MW (MW) is determined. w ), number average MW(M n ), MW corresponding to the maximum value of the chromatographic peak (M p ), z-mean MW(M z or M z+1 ) or viscosity average MW (M v ) in one or more (e.g., 2, 3, 4, or 5). In some embodiments, the polydispersity index is determined, for example, by calculating M w With M n In some embodiments, determining the R of LNP gw, for example, using Zimm or partial Zimm methods (e.g., as described in Wyatt.AnalyticaChimicaActa.1993.272:1-40). In some embodiments, the half-life of the LNP is determined. In some embodiments, the stability of the LNP is determined according to the methods described herein, for example, the methods described in Example 1.

[0060] In some embodiments, the method comprises directly comparing the obtained MALS signals. In some embodiments, the method comprises indirectly comparing the obtained MALS signals.

[0061] In some embodiments, a plurality of LNPs in the sample are analyzed, eg, according to the methods described herein.

[0062] In some embodiments, the method does not include the step of recovering LNP, for example, by ultracentrifugation. In some embodiments, the method does not include the step of diluting the sample, for example, removing the high molecular weight components (for example, plasma or serum components) that interfere with detection (for example, by DLS detection). In some embodiments, the method does not include the step of labeling LNP, for example, the step of labeling LNP with a fluorophore. In some embodiments, the method quantitatively detects the LNP in the sample.

[0063] In some embodiments, LNP loads nucleic acid. In some embodiments, LNP does not load nucleic acid. In some embodiments, the nucleic acid is a therapeutic nucleic acid (TNA). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a vaccine. In some embodiments, the nucleic acid is a non-coding RNA, for example, a small non-coding RNA. In some embodiments, the nucleic acid is a small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or a micro RNA (miRNA). In some embodiments, the nucleic acid is a guide RNA (gRNA). In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid includes one or more modified nucleotides. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded.

[0064] In some embodiments, the sample comprises a physiological fluid. In some embodiments, the physiological fluid is plasma. In some embodiments, the physiological fluid is serum. In some embodiments, the physiological fluid is blood. In some embodiments, the physiological fluid is amniotic fluid, aqueous humor, bile, breast milk, cerebrospinal fluid, cerumen, chyle, exudate, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural effusion, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine or vomit. In some embodiments, the physiological fluid has a pH of 2-10, for example, 3-9, 4-8, 5-7, 2-8, 2-6, 2-4, 8-10, 6-10, 4-10, 2-10, 2-4, 3-5, 4-6, 6-8 or 7-9 pH. In some embodiments, the physiological fluid has the pH of serum or plasma (for example, 7.3-7.5). In some embodiments, the physiological fluid has the pH of a tumor microenvironment (eg, pH 5.6 to 6.8).

[0065] In another aspect, the present disclosure provides a method for monitoring the preparation process of lipid nanoparticles (LNPs). The method comprises: (optionally) obtaining a first sample containing LNPs from the process of preparing LNPs; performing size exclusion chromatography (SEC) on the first sample; and obtaining a multi-angle light scattering (MALS) signal from the first sample to monitor the preparation process of LNPs.

[0066] In some embodiments, the method further comprises: (optionally) obtaining a second sample comprising LNPs from the process of preparing LNPs; performing size exclusion chromatography (SEC) on the second sample; and obtaining a second MALS signal from the second sample. In some embodiments, the second sample is obtained after the first sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the first sample is obtained.

[0067] In some embodiments, the method further comprises: (optionally) obtaining a third sample comprising LNPs from the process of preparing LNPs; performing size exclusion chromatography (SEC) on the third sample; and obtaining a third MALS signal from the third sample. In some embodiments, the third sample is obtained after obtaining the second sample, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after obtaining the second sample.

[0068] In some embodiments, the method further comprises: (optionally) obtaining a fourth sample comprising LNPs from the process of preparing LNPs; performing size exclusion chromatography (SEC) on the fourth sample; and obtaining a fourth MALS signal from the fourth sample. In some embodiments, the fourth sample is obtained after the third sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the third sample is obtained.

[0069] In some embodiments, the method further comprises: (optionally) obtaining a fifth sample comprising LNPs from the process of preparing LNPs; performing size exclusion chromatography (SEC) on the fifth sample; and obtaining a fifth MALS signal from the fifth sample. In some embodiments, the fifth sample is obtained after obtaining the fourth sample, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after obtaining the fourth sample.

[0070] In some embodiments, SEC is carried out in a single column configuration. In some embodiments, SEC is carried out in a dual column configuration, for example, using two columns with different pore sizes, for example, a first column with a first average pore size and a second column with a second average pore size. In some embodiments, the first average pore size is greater than the second average pore size. In some embodiments, the first average pore size has a molecular weight cutoff (MWCO) of about 1 MDa or more, for example, about 2 MDa or more, 5 MDa or more, 10 MDa or more, 15 MDa or more or 20 MDa or more. In some embodiments, the first average pore size has a MWCO of about 20 MDa or less, for example, about 15 MDa or less, 10 MDa or less, 5 MDa or less, 2 MDa or less or 1 MDa or less. In some embodiments, the first average pore size has a MWCO of about 1 MDa to 20 MDa, for example, about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, for example, about 10 MDa. In some embodiments, the second average pore size has a MWCO of about 10 MDa or less, for example, about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less, or 0.1 MDa or less. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa or greater, e.g., about 0.2 MDa or greater, 0.5 MDa or greater, 1 MDa or greater, 2 MDa or greater, 5 MDa or greater, or 10 MDa or greater. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa to 10 MDa, for example, about 0.2 MDa to 5 MDa, 0.5 MDa to 2 MDa, 0.1 MDa to 5 MDa, 0.1 MDa to 2 MDa, 0.1 MDa to 1 MDa, 0.1 MDa to 0.5 MDa, 0.1 MDa to 0.2 MDa, 5 MDa to 10 MDa, 2 MDa to 10 MDa, 1 MDa to 10 MDa, 0.5 MDa to 10 MDa, 0.2 MDa to 10 MDa, 0.2 MDa to 1 MDa, 1 MDa to 5 MDa, or 0.3 MDa to 0.7 MDa, for example, about 0.5 MDa.

[0071] In some embodiments, SEC is performed using a column having a length of 100 mm or more, e.g., about 150 mm or more, 200 mm or more, 250 mm or more, 300 mm or more, 400 mm or more, 500 mm or more, 600 mm or more, 700 mm or more, 800 mm or more, 900 mm or more, or 1000 mm or more, e.g., about 100 mm to 1000 mm, 150 mm to 500 mm, or about 200 mm to 300 mm, e.g., about 250 mm.

[0072] In some embodiments, SEC is performed using a polymer matrix column. In some embodiments, SEC is not performed using a silica matrix column.

[0073] In some embodiments, SEC is performed under aqueous conditions, e.g., using a biocompatible buffer system, e.g., phosphate buffer, bicarbonate buffer, acetate buffer, Tris buffer, or Good's buffer (e.g., as described in Good et al. Biochemistry. 1966; 5(2):467-77; Good and Izawa Methods Enzymol. 1972; 24:53-68; Ferguson et al. Anal. Biochem. 1980 May; 104 (2): 300-10) any one, for example, MES, Bis-tris methane, ADA, Bis-tris propane, PIPES, ACES, MOPSO, choline chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetylaminoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tris (hydroxymethyl) methylglycine (Tricine), tris hydroxymethylaminomethane (Tris), glycine amide, glycine peptide, HEPBS, N-bihydroxyethylglycine (Bicine), TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS or any one of a combination thereof. In some embodiments, SEC is performed without using an organic solvent (e.g., THF) as the mobile phase.

[0074] In some embodiments, MALS is performed at scattering angles of 15° and 90°. In some embodiments, MALS is performed using a laser wavelength of about 500nm to 800nm, e.g., about 600nm to 700nm, 500nm to 700nm, 600nm to 800nm, 500nm to 600nm, 700nm to 800nm, e.g., about 658nm. In some embodiments, MALS is performed using a sample cell volume of about 1 μL to 50 μL, e.g., about 2 μL to 25 μL, 5 μL to 20 μL, 1 μL to 40 μL, 1 μL to 30 μL, 1 μL to 20 μL, 1 μL to 10 μL, 40 μL to 50 μL, 30 μL to 50 μL, 20 μL to 50 μL, 10 μL to 50 μL, 5 μL to 15 μL, or 10 μL to 20 μL, e.g., about 10 μL. In some embodiments, MALS is performed using a scattering volume of about 0.001 μL to 0.1 μL, e.g., about 0.005 μL to 0.05 μL, 0.001 μL to 0.05 μL, 0.001 μL to 0.01 μL, 0.05 μL to 0.1 μL, 0.01 μL to 0.1 μL, 0.005 μL to 0.1 μL, or 0.005 μL to 0.05 μL, e.g., about 0.01 μL. In some embodiments, MALS is performed at a temperature range of about 20°C to 70°C, e.g., about 25°C to 75°C or 30°C to 60°C. In some embodiments, MALS is performed with a temperature stability of no more than ±1°C, e.g., no more than ±0.5°C or ±0.2°C. In some embodiments, MALS is performed at a pH range of about 1-12, e.g., about 2-11 or 2-10.

[0075] In some embodiments, the size of the LNP is determined, for example, the hydrodynamic radius (R h In some embodiments, the molecular weight (MW) of the LNP is determined, for example, the weight average MW (MW) is determined. w ), number average MW(M n ), MW corresponding to the maximum value of the chromatographic peak (M p ), z-mean MW(M z or M z+1 ) or viscosity average MW (M v ) in one or more (e.g., 2, 3, 4, or 5). In some embodiments, the polydispersity index is determined, for example, by calculating M w With M n In some embodiments, determining the R of LNP gw, for example, using Zimm or partial Zimm methods (e.g., as described in Wyatt.AnalyticaChimicaActa.1993.272:1-40). In some embodiments, the half-life of the LNP is determined. In some embodiments, the stability of the LNP is determined according to the methods described herein, for example, the methods described in Example 1.

[0076] In some embodiments, the method comprises directly comparing the obtained MALS signals. In some embodiments, the method comprises indirectly comparing the obtained MALS signals.

[0077] In some embodiments, a plurality of LNPs in the sample are analyzed, eg, according to the methods described herein.

[0078] In some embodiments, the method does not include the step of recovering LNP, for example, by ultracentrifugation. In some embodiments, the method does not include the step of diluting the sample, for example, removing the high molecular weight components (for example, plasma or serum components) that interfere with detection (for example, by DLS detection). In some embodiments, the method does not include the step of labeling LNP, for example, the step of labeling LNP with a fluorophore. In some embodiments, the method quantitatively detects the LNP in the sample.

[0079] In some embodiments, LNP loads nucleic acid. In some embodiments, LNP does not load nucleic acid. In some embodiments, the nucleic acid is a therapeutic nucleic acid (TNA). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a vaccine. In some embodiments, the nucleic acid is a non-coding RNA, for example, a small non-coding RNA. In some embodiments, the nucleic acid is a small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or a micro RNA (miRNA). In some embodiments, the nucleic acid is a guide RNA (gRNA). In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid includes one or more modified nucleotides. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded.

[0080] In some embodiments, the sample comprises a physiological fluid. In some embodiments, the physiological fluid is plasma. In some embodiments, the physiological fluid is serum. In some embodiments, the physiological fluid is blood. In some embodiments, the physiological fluid is amniotic fluid, aqueous humor, bile, breast milk, cerebrospinal fluid, cerumen, chyle, exudate, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural effusion, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine or vomit. In some embodiments, the physiological fluid has a pH of 2-10, for example, 3-9, 4-8, 5-7, 2-8, 2-6, 2-4, 8-10, 6-10, 4-10, 2-10, 2-4, 3-5, 4-6, 6-8 or 7-9 pH. In some embodiments, the physiological fluid has the pH of serum or plasma (for example, 7.3-7.5). In some embodiments, the physiological fluid has the pH of a tumor microenvironment (eg, pH 5.6 to 6.8).

[0081] In another aspect, the present disclosure provides a method for determining the pharmacokinetics (PK) of a lipid nanoparticle (LNP). The method comprises: (optionally) obtaining a first sample comprising LNP from a subject; performing size exclusion chromatography (SEC) on the first sample; and obtaining a multi-angle light scattering (MALS) signal from the first sample to determine the PK of the LNP.

[0082] In some embodiments, the method further comprises: (optionally) obtaining a second sample comprising LNPs from the subject; performing size exclusion chromatography (SEC) on the second sample; and obtaining a second MALS signal from the second sample. In some embodiments, the second sample is obtained after the first sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the first sample is obtained.

[0083] In some embodiments, the method further comprises: (optionally) obtaining a third sample comprising LNPs from the subject; performing size exclusion chromatography (SEC) on the third sample; and obtaining a third MALS signal from the third sample. In some embodiments, the third sample is obtained after the second sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the second sample is obtained.

[0084] In some embodiments, the method further comprises: (optionally) obtaining a fourth sample comprising LNPs from the subject; performing size exclusion chromatography (SEC) on the fourth sample; and obtaining a fourth MALS signal from the fourth sample. In some embodiments, the fourth sample is obtained after the third sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after the third sample is obtained.

[0085] In some embodiments, the method further comprises: (optionally) obtaining a fifth sample comprising LNPs from the subject; performing size exclusion chromatography (SEC) on the fifth sample; and obtaining a fifth MALS signal from the fifth sample. In some embodiments, the fifth sample is obtained after obtaining the fourth sample, for example, at least 15, 30, 45, 60, 75, 90, 105, or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours after obtaining the fourth sample.

[0086] In some embodiments, the subject is a healthy subject. In some embodiments, the subject suffers from or may suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder. In some embodiments, the subject is a human. In some embodiments, the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0087] In some embodiments, SEC is carried out in a single column configuration. In some embodiments, SEC is carried out in a dual column configuration, for example, using two columns with different pore sizes, for example, a first column with a first average pore size and a second column with a second average pore size. In some embodiments, the first average pore size is greater than the second average pore size. In some embodiments, the first average pore size has a molecular weight cutoff (MWCO) of about 1 MDa or more, for example, about 2 MDa or more, 5 MDa or more, 10 MDa or more, 15 MDa or more or 20 MDa or more. In some embodiments, the first average pore size has a MWCO of about 20 MDa or less, for example, about 15 MDa or less, 10 MDa or less, 5 MDa or less, 2 MDa or less or 1 MDa or less. In some embodiments, the first average pore size has a MWCO of about 1 MDa to 20 MDa, for example, about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, for example, about 10 MDa. In some embodiments, the second average pore size has a MWCO of about 10 MDa or less, for example, about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less, or 0.1 MDa or less. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa or greater, e.g., about 0.2 MDa or greater, 0.5 MDa or greater, 1 MDa or greater, 2 MDa or greater, 5 MDa or greater, or 10 MDa or greater. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa to 10 MDa, for example, about 0.2 MDa to 5 MDa, 0.5 MDa to 2 MDa, 0.1 MDa to 5 MDa, 0.1 MDa to 2 MDa, 0.1 MDa to 1 MDa, 0.1 MDa to 0.5 MDa, 0.1 MDa to 0.2 MDa, 5 MDa to 10 MDa, 2 MDa to 10 MDa, 1 MDa to 10 MDa, 0.5 MDa to 10 MDa, 0.2 MDa to 10 MDa, 0.2 MDa to 1 MDa, 1 MDa to 5 MDa, or 0.3 MDa to 0.7 MDa, for example, about 0.5 MDa.

[0088] In some embodiments, SEC is performed using a column having a length of 100 mm or more, e.g., about 150 mm or more, 200 mm or more, 250 mm or more, 300 mm or more, 400 mm or more, 500 mm or more, 600 mm or more, 700 mm or more, 800 mm or more, 900 mm or more, or 1000 mm or more, e.g., about 100 mm to 1000 mm, 150 mm to 500 mm, or about 200 mm to 300 mm, e.g., about 250 mm.

[0089] In some embodiments, SEC is performed using a polymer matrix column. In some embodiments, SEC is not performed using a silica matrix column.

[0090] In some embodiments, SEC is performed under aqueous conditions, e.g., using a biocompatible buffer system, e.g., phosphate buffer, bicarbonate buffer, acetate buffer, Tris buffer, or Good's buffer (e.g., as described in Good et al. Biochemistry. 1966; 5(2):467-77; Good and Izawa Methods Enzymol. 1972; 24:53-68; Ferguson et al. Anal. Biochem. 1980 May; 104 (2): 300-10) any one, for example, MES, Bis-tris methane, ADA, Bis-tris propane, PIPES, ACES, MOPSO, choline chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetylaminoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tris (hydroxymethyl) methylglycine (Tricine), tris hydroxymethylaminomethane (Tris), glycine amide, glycine peptide, HEPBS, N-bihydroxyethylglycine (Bicine), TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS or any one of a combination thereof. In some embodiments, SEC is performed without using an organic solvent (e.g., THF) as the mobile phase.

[0091] In some embodiments, MALS is performed at scattering angles of 15° and 90°. In some embodiments, MALS is performed using a laser wavelength of about 500nm to 800nm, e.g., about 600nm to 700nm, 500nm to 700nm, 600nm to 800nm, 500nm to 600nm, 700nm to 800nm, e.g., about 658nm. In some embodiments, MALS is performed using a sample cell volume of about 1 μL to 50 μL, e.g., about 2 μL to 25 μL, 5 μL to 20 μL, 1 μL to 40 μL, 1 μL to 30 μL, 1 μL to 20 μL, 1 μL to 10 μL, 40 μL to 50 μL, 30 μL to 50 μL, 20 μL to 50 μL, 10 μL to 50 μL, 5 μL to 15 μL, or 10 μL to 20 μL, e.g., about 10 μL. In some embodiments, MALS is performed using a scattering volume of about 0.001 μL to 0.1 μL, e.g., about 0.005 μL to 0.05 μL, 0.001 μL to 0.05 μL, 0.001 μL to 0.01 μL, 0.05 μL to 0.1 μL, 0.01 μL to 0.1 μL, 0.005 μL to 0.1 μL, or 0.005 μL to 0.05 μL, e.g., about 0.01 μL. In some embodiments, MALS is performed at a temperature range of about 20°C to 70°C, e.g., about 25°C to 75°C or 30°C to 60°C. In some embodiments, MALS is performed with a temperature stability of no more than ±1°C, e.g., no more than ±0.5°C or ±0.2°C. In some embodiments, MALS is performed at a pH range of about 1-12, e.g., about 2-11 or 2-10.

[0092] In some embodiments, the size of the LNP is determined, for example, the hydrodynamic radius (R h In some embodiments, the molecular weight (MW) of the LNP is determined, for example, the weight average MW (MW) is determined. w ), number average MW(M n ), MW corresponding to the maximum value of the chromatographic peak (M p ), z-mean MW(M z or M z+1 ) or viscosity average MW (M v ) in one or more (e.g., 2, 3, 4, or 5). In some embodiments, the polydispersity index is determined, for example, by calculating M w With M n In some embodiments, determining the R of LNP gw, for example, using Zimm or partial Zimm methods (e.g., as described in Wyatt.AnalyticaChimicaActa.1993.272:1-40). In some embodiments, the half-life of the LNP is determined. In some embodiments, the stability of the LNP is determined according to the methods described herein, for example, the methods described in Example 1.

[0093] In some embodiments, the method comprises directly comparing the obtained MALS signals. In some embodiments, the method comprises indirectly comparing the obtained MALS signals.

[0094] In some embodiments, a plurality of LNPs in the sample are analyzed, eg, according to the methods described herein.

[0095] In some embodiments, the method does not include the step of recovering LNP, for example, by ultracentrifugation. In some embodiments, the method does not include the step of diluting the sample, for example, removing the high molecular weight components (for example, plasma or serum components) that interfere with detection (for example, by DLS detection). In some embodiments, the method does not include the step of labeling LNP, for example, the step of labeling LNP with a fluorophore. In some embodiments, the method quantitatively detects the LNP in the sample.

[0096] In some embodiments, LNP loads nucleic acid. In some embodiments, LNP does not load nucleic acid. In some embodiments, the nucleic acid is a therapeutic nucleic acid (TNA). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a vaccine. In some embodiments, the nucleic acid is a non-coding RNA, for example, a small non-coding RNA. In some embodiments, the nucleic acid is a small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or a micro RNA (miRNA). In some embodiments, the nucleic acid is a guide RNA (gRNA). In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid includes one or more modified nucleotides. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded.

[0097] In some embodiments, the sample comprises a physiological fluid. In some embodiments, the physiological fluid is plasma. In some embodiments, the physiological fluid is serum. In some embodiments, the physiological fluid is blood. In some embodiments, the physiological fluid is amniotic fluid, aqueous humor, bile, breast milk, cerebrospinal fluid, cerumen, chyle, exudate, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural effusion, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine or vomit. In some embodiments, the physiological fluid has a pH of 2-10, for example, 3-9, 4-8, 5-7, 2-8, 2-6, 2-4, 8-10, 6-10, 4-10, 2-10, 2-4, 3-5, 4-6, 6-8 or 7-9 pH. In some embodiments, the physiological fluid has the pH of serum or plasma (for example, 7.3-7.5). In some embodiments, the physiological fluid has the pH of a tumor microenvironment (eg, pH 5.6 to 6.8).

[0098] In another aspect, the present disclosure provides a system comprising a processor configured to perform the method as described herein.

[0099] In some embodiments, the processor is configured to perform size exclusion chromatography (SEC) on the sample or an aliquot of the sample. In some embodiments, the processor is further configured to obtain a multi-angle light scattering signal (MALS) from the sample or an aliquot of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1A Schematic diagram depicting an exemplary mRNA-LNP detection using the SEC-MALS pipeline.

[0101] Figure 1B Figure 2 is a SEC-MALS chromatogram of mRNA-LNP in 50 mM phosphate buffer (pH 7.2). The mRNA-LNP peak is marked as "1" at the bottom. Dissolved air and buffer salts produce artifacts in the RI trace, which are well separated from the mRNA-LNP peak and do not affect the calculated M w and R gw value.

[0102] Figures 2A-2D Describes the separation of human plasma from mRNA-LNPs by dual-column SEC. LS90°( Figure 2A-2B ) and refractive index ( Figure 2C-2D ) Chromatogram showing minimal interference of plasma components on the mRNA-LNP peak in the dual-column configuration.

[0103] Figures 3A-3DDegradation of mRNA-LNP in human plasma is described. The LS90°( Figure 3A ), UV absorbance at 260 nm ( Figure 3B ) and refractive index ( Figure 3C ) Chromatogram. 30 μl injection was performed every 15 min. Figure 3B-3C The inset shows a zoomed-in representation of the mRNA-LNP peak. The superimposed online dynamic light scattering signals of all injections are shown in Figure 3D .

[0104] Figures 4A-4D Describe the degradation kinetics and physical characteristics of LNPs revealed by MALS. The degradation kinetics of mRNA-LNPs and empty LNPs in human plasma and serum traced by LS 90° peak area are shown in Figure 4A . Figure 4B showed that mRNA-LNPs maintained a constant apparent Mw in serum but not in plasma. Figure 4C-4D showed that mRNA-LNPs maintained a constant Rgw in serum but not in plasma.

[0105] Figures 5A-5C LC-MS / MS quantification of mRNA-LNP composition during degradation in serum and plasma. Overlaid PRM chromatograms of cholesterol (369.65→147.16), DSPC (790.82→184.13), ALC-0315 (766.91→510.62), and ALC-0159 (1184.10→494.62) are shown in Figure 5A Quantitative injection: 1, 2, 4 and 8 pmol DSPC, ALC-0315 and ALC-0159; 10, 20, 40 and 80 pmol cholesterol. For the mass spectra of each compound, see Figures 9A-9D The compositions of degraded mRNA-LNPs in serum and plasma are shown in Figure 5B-5C .

[0106] Figures 6A-6D Describes the effect of lipid impurities in ionizable lipids on the stability of mRNA-LNP in serum. Fig. 6A ) and impurities ( Figure 6B ) of the total ion chromatogram of the lipid component from mRNA-LNP. The impurity peak eluted at 6.5 min and is marked with a red triangle. Cholesterol is shown in the extracted ion chromatogram shown in the inset. The mass spectrum of the impurity peak with isotopic resolution shown in the inset is shown in Figure 6C The degradation of mRNA-LNPs in serum with and without impurities is shown in Fig.6D .

[0107] Figure 7A-7BDescribe the molecular structure of ALC-0315 ( Fig. 7A ) and the assumed molecular structure of the impurity ( Figure 7B ). Theoretical monoisotopic masses are shown.

[0108] Figure 8 Depicting an ALC-0315 impurity containing an O-Boc protecting group. Shown are extracted ion chromatograms of ALC-0315 without impurity (top), ALC-0315 impurity fraction (middle), deprotected ALC-0315 impurity fraction (bottom), m / z=766.72 and 866.82. Low sample pH causes a slight retention time shift in the graph (bottom).

[0109] Figures 9A-9D Describe cholesterol ( Fig. 9A )、DSPC( Fig. 9B )、ALC-0315( Fig. 9C ) and ALC-0159( Fig.9D ) MS / MS spectrum. The MS1 spectrum is shown in the inset ( Fig. 9B ,9D), where the precursor ion is not obvious in the MS / MS spectrum.

[0110] Figure 10-10D shows cholesterol ( Fig. 10A )、DSPC( Fig. 10B )、ALC-0315( Fig. 10C ) and ALC-0159( Fig. 10D ) representative calibration curve.

[0111] Fig.11A The LC-UV chromatogram of an impure batch of ALC-0315. The absorbance at 215 nm was used to monitor the ester groups in the eluted compounds and for the relative quantification of ALC-0315 (RT = 15.48 min) and contaminants (RT = 17.66–24.86 min). Fig. 11B MS1 depicting the ALC-0315 peak, showing only a trace amount of an impurity (866.9 m / z, red arrow). Fig. 11C MS1 depicting the contaminant, showing a stronger impurity signal (866.9 m / z, red arrow).

[0112] Fig. 12A The LC-UV chromatogram of an impure batch of ALC-0315. The absorbance at 215 nm was used to monitor the ester groups in the eluted compounds and for the relative quantification of ALC-0315 (RT = 14.96 min) and contaminants (RT = 17.60–25.16 min). Fig. 12B Describe the MS1 of the ALC-0315 peak. Fig. 12C Describe the MS1 of the pollutant. The main peak ( Fig. 12B ) and impurities ( Fig. 12C ) showed no obvious impurity signal at 866.9 m / z in MS1. DETAILED DESCRIPTION

[0113] The present disclosure is based at least in part on the discovery that size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) is a highly effective tool for quantitatively characterizing LNP stability in physiological fluids. The optimized chromatography allows separation of LNPs from interfering plasma and serum components, allowing accurate characterization of reaction kinetics and changes in nanoparticle physical properties.

[0114] Many emerging LNP drugs may require systemic administration of LNPs rather than more local intramuscular injection. Since intravenous injection brings LNPs into contact with human blood, the stability of LNPs in human plasma or serum is an important factor in drug development as it is expected to affect efficacy and pharmacokinetics.

[0115] However, the method for measuring the stability of LNPs after being exposed to human plasma or serum has some defects at present. Most methods rely on dynamic light scattering (DLS) to measure the LNP size distribution after being recovered by ultracentrifugation or gel filtration to establish stability. In some cases, DLS is used to directly measure the size distribution of LNPs in dilute serum solutions. Unfortunately, DLS can not accurately determine the quantity of complete LNPs in a period of time, which is the most direct measurement standard of stability. In general, DLS can only measure size distribution and can not measure the quantity of particles of any given size.

[0116] Specifically, if almost all of the recovered LNPs are close to the original size, the DLS-based method may consider the LNP sample to be stable even if fewer LNPs are recovered due to an ongoing degradation process. In principle, an increase in the polydispersity of LNPs may indicate degradation, but in practice this is often not the case due to the greater buoyancy or smaller hydrodynamic radius (R) of the LNPs. h ), ultracentrifugation or gel filtration cannot effectively recover partially degraded LNPs, which may cause confusion.

[0117] Furthermore, LNP composition can be altered by plasma components, although it may retain its original R h , but expectations may change their properties. Unfortunately, DLS provides little insight into these processes.

[0118] The method described herein uses size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) to quantitatively study the stability of widely used mRNA-LNP formulations in the presence of human plasma or serum. By separating the mRNA-LNP from the interfering plasma and serum components, the changes in the reaction kinetics and the physical properties of the nanoparticles can be accurately characterized. In addition, the influence of trace contaminants in lipid excipients on the stability of mRNA-LNP can be studied.

[0119] The present disclosure provides methods with improved accuracy for measuring the stability of LNPs in physiological fluids. After separation of LNPs from interfering plasma or serum components by SEC, MALS can be used to measure the number of intact LNPs remaining with high sensitivity based on their retention time. The degradation kinetics depends on the environment (purified serum albumin vs. plasma / serum) and the characteristics of the LNPs (containing mRNA vs. empty). Based on the measured biophysical parameters (R gw and apparent M w ), SEC-MALS can reveal that LNP lipids are gradually replaced by plasma components (probably proteins), which can be confirmed by LC-MS / MS. Therefore, the methods described herein can be used to optimize LNP formulations. The methods described herein can also detect the destabilization of mRNA-LNPs caused by impurities, demonstrating its practicality as a quality control tool.

[0120] definition

[0121] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0122] As used herein, the articles "a" and "an" refer to one or to more than one (eg, to at least one) of the grammatical object of the article.

[0123] As used herein, the terms "about" and "approximately" generally mean an acceptable degree of error for the amount measured, given the nature or precision of the measurement. An exemplary degree of error is within 20 percent (%) of a given value or range of values, typically within 10%, and more typically within 5%.

[0124] As used herein, the terms "Acquire" or "acquiring" refer to obtaining possession of a physical entity or value (e.g., a numerical value) by "directly obtaining" or "indirectly obtaining" the physical entity or value. "Directly obtaining" means performing a process (e.g., performing a synthesis or analytical method) to obtain the physical entity or value. "Indirectly obtaining" refers to receiving the entity or value from another party or source (e.g., a third-party laboratory that directly obtains the physical entity or value). Directly obtaining a physical entity includes performing a process that includes physical changes to a physical substance (e.g., a starting material). Exemplary changes include preparing a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, and performing a chemical reaction that includes breaking or forming covalent or non-covalent bonds. Directly obtaining a value includes performing a process that includes a physical change in a sample or other substance, for example, performing an analytical process (sometimes referred to herein as "physical analysis") that includes a physical change in a substance (e.g., a sample, an analyte, or a reagent), performing an analytical method, for example, a method that includes one or more of the following: separating or purifying a substance, for example, an analyte or a fragment or other derivative thereof, from another substance; combining an analyte or a fragment or other derivative thereof with another substance, such as a buffer, a solvent, or a reactant; or changing the structure of an analyte or a fragment or other derivative thereof, for example, by breaking or forming a covalent or non-covalent bond between a first and a second atom of the analyte; or by changing the structure of a reagent or a fragment or other derivative thereof, for example, by breaking or forming a covalent or non-covalent bond between a first and a second atom of the reagent. In one embodiment, directly obtaining encompasses direct measurement. In one embodiment, indirectly obtaining encompasses inference.

[0125] As used herein, the term "obtaining a sample" refers to obtaining possession of the sample, either by "directly obtaining" or "indirectly obtaining" the sample (e.g., a sample as described herein). "Directly obtaining a sample" means performing a process (e.g., performing a physical method such as surgery or extraction) to obtain the sample. "Indirectly obtaining a sample" refers to receiving a sample from another party or source (e.g., a third-party laboratory that directly obtains the sample). Directly obtaining a sample includes performing a process that includes a physical change in a physical substance, for example, a starting material such as a tissue, such as tissue in a human patient or tissue previously isolated from a patient. Exemplary changes include preparing a physical entity from a starting material; dissecting or scraping a tissue; separating or purifying a substance; combining two or more separate entities into a mixture; or performing a chemical reaction that includes breaking or forming covalent or non-covalent bonds.

[0126] Unless the context clearly indicates otherwise, the use of "or" in this document is intended to mean the term "and / or" and is used interchangeably with the term "and / or". Unless the context clearly indicates otherwise, the use of the term "and / or" in certain places in this document does not mean that the use of the term "or" cannot be interchangeable with the term "and / or".

[0127] As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest. In one embodiment, the source of interest includes an organism, such as an animal or a human. The source of the sample can be blood or blood components; body fluid; solid tissue from fresh, frozen and / or preserved organs, tissues, biopsies (biopsy), resections (resectionor), smears (smear) or aspirates (aspirate); or cells from any period in the embryo or embryonic development of a subject. In one embodiment, the source of the sample is blood or blood components. In one embodiment, the sample is an original sample, for example, obtained directly from a source of interest by any appropriate means. In one embodiment, the sample is a product obtained by processing the original sample (for example, by removing one or more components and / or by adding one or more reagents).

[0128] The term "subject" as used herein is intended to include humans and non-human animals. In some embodiments, the subject is a human subject, e.g., a healthy human subject or a human subject suffering from a disease or at risk of suffering from a disease. The term "non-human animal" includes mammals and non-mammals, such as non-human primates.

[0129] Size Exclusion Chromatography (SEC)

[0130] The methods or systems described herein may include size exclusion chromatography (SEC).

[0131] SEC, sometimes referred to as molecular sieve chromatography, is a chromatographic method in which molecules in solution are usually separated by their size. Typically, when an aqueous solution is used to transport a sample through a chromatographic column, the technique is called gel filtration chromatography. It uses spherical beads containing pores of a specific size distribution. Separation usually occurs when molecules of different sizes are included or excluded from the pores within the matrix. Smaller molecules diffuse into the pores and, depending on their size, their flow through the column is hindered, while larger molecules do not enter the pores and elute in the void volume of the column. Therefore, the molecules are separated based on their size as they pass through the chromatographic column and elute in order of decreasing molecular weight (MW).

[0132] SEC can be performed in a single column configuration or a multi-column configuration, for example, using multiple columns with different pore sizes. In some embodiments, SEC is performed in a dual column configuration, for example, using two columns with different pore sizes. For example, SEC can be performed using a first column having a first average pore size and a second column having a second average pore size.

[0133] In some embodiments, the first average pore size is greater than the second average pore size. In some embodiments, the first average pore size has a molecular weight cutoff (MWCO) of about 1 MDa or more, for example, about 2 MDa or more, 5 MDa or more, 10 MDa or more, 15 MDa or more, or 20 MDa or more. In some embodiments, the first average pore size has a MWCO of about 20 MDa or less, for example, about 15 MDa or less, 10 MDa or less, 5 MDa or less, 2 MDa or less, or 1 MDa or less. In some embodiments, the first average pore size has a MWCO of about 1 MDa to 20 MDa, for example, about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, for example, about 10 MDa.

[0134] In some embodiments, the second average pore size has a MWCO of about 10 MDa or less, for example, about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less, or 0.1 MDa or less. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa or greater, for example, about 0.2 MDa or greater, 0.5 MDa or greater, 1 MDa or greater, 2 MDa or greater, 5 MDa or greater, or 10 MDa or greater. In some embodiments, the second average pore size has a MWCO of about 0.1 MDa to 10 MDa, for example, about 0.2 MDa to 5 MDa, 0.5 MDa to 2 MDa, 0.1 MDa to 5 MDa, 0.1 MDa to 2 MDa, 0.1 MDa to 1 MDa, 0.1 MDa to 0.5 MDa, 0.1 MDa to 0.2 MDa, 5 MDa to 10 MDa, 2 MDa to 10 MDa, 1 MDa to 10 MDa, 0.5 MDa to 10 MDa, 0.2 MDa to 10 MDa, 0.2 MDa to 1 MDa, 1 MDa to 5 MDa, or 0.3 MDa to 0.7 MDa, for example, about 0.5 MDa.

[0135] In some embodiments, the first average pore size has a MWCO of about 1 MDa or greater (e.g., about 2 MDa or greater, 5 MDa or greater, 10 MDa or greater, 15 MDa or greater, or 20 MDa or greater) and the second average pore size has a MWCO of about 10 MDa or less (e.g., about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less, or 0.1 MDa or less), and the first average pore size is larger than the second average pore size. In some embodiments, the first average pore size has a MWCO of about 1 MDa to 20 MDa (e.g., about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, e.g., about 10 MDa) and the second average pore size has a MWCO of about 0.1 MDa to 10 MDa (e.g., About 0.2MDa to 5MDa, 0.5MDa to 2MDa, 0.1MDa to 5MDa, 0.1MDa to 2MDa, 0.1MDa to 1MDa, 0.1MDa to 0.5MDa, 0.1MDa to 0.2MDa, 5MDa to 10MDa, 2MDa to 10MDa, 1MDa to 10MDa, 0.5MDa to 10MDa, 0.2MDa to 10MDa, 0.2MDa to 1MDa, 1MDa to 5MDa or 0.3MDa to 0.7MDa, for example, about 0.5MDa) of the MWCO, and the first average pore size is greater than the second average pore size.

[0136] In some embodiments, SEC is performed using a column having a length of 100 mm or more, e.g., about 150 mm or more, 200 mm or more, 250 mm or more, 300 mm or more, 400 mm or more, 500 mm or more, 600 mm or more, 700 mm or more, 800 mm or more, 900 mm or more, or 1000 mm or more, e.g., about 100 mm to 1000 mm, 150 mm to 500 mm, or about 200 mm to 300 mm, e.g., about 200 mm, 250 mm, 300 mm, 400 mm, 500 mm, 600 mm, 700 mm, 800 mm, 900 mm, or 1000 mm.

[0137] In some embodiments, SEC is performed using a polymer matrix column. In some embodiments, SEC is performed without a polymer matrix column. In some embodiments, SEC is performed without a silica matrix column. In some embodiments, SEC is performed using a silica matrix column.

[0138] In some embodiments, SEC is performed under aqueous conditions, for example, using a biocompatible buffer system. Exemplary buffers include, but are not limited to, phosphate buffer, bicarbonate buffer, or Tris buffer. Other exemplary buffers include any of Good's buffers, as described in Good et al. Biochemistry. 1966; 5(2): 467-77; Good and Izawa Methods Enzymol. 1972; 24: 53-68; Ferguson et al. Anal Biochem. 1980; 104(2): 300-10. For example, the buffer can be any one of MES, Bis-tris methane, ADA, Bis-tris propane, PIPES, ACES, MOPSO, cholestyramine chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetylaminoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tris(hydroxymethyl)methylglycine (Tricine), tris(hydroxymethyl)aminomethane (Tris), glycineamide, glycine, HEPBS, Bicine, TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS or a combination thereof.

[0139] In some embodiments, SEC is performed without using an organic solvent (eg, tetrahydrofuran or THF) as the mobile phase.

[0140] Multi-angle light scattering (MALS)

[0141] The methods and systems described herein may use multi-angle light scattering.

[0142] Multi angle light scattering (MALS), sometimes referred to as multiangle light scattering or multi-angle light scattering, is a technique for measuring light scattered by a sample at multiple angles. It can be used to determine the absolute molar mass and average size of molecules in solution by detecting the light scattered by the molecules. Most commonly used is a collimated beam from a laser source, in which case the technique may be referred to as multi-angle laser light scattering (MALLS). The term "multi-angle" refers to detecting scattered light at different discrete angles, for example, by a single detector that moves within a range that includes a selected specific angle or by an array of detectors that are fixed at specific angular positions.

[0143] In some embodiments, MALS is performed at two scattering angles that differ by 15°, 30°, 45°, 60°, 75°, or 90°. In some embodiments, MALS is performed at scattering angles of about 15° and about 90°. In some embodiments, MALS is performed using a laser wavelength of about 500nm to 800nm, e.g., about 600nm to 700nm, 500nm to 700nm, 600nm to 800nm, 500nm to 600nm, 700nm to 800nm, e.g., about 658nm. In some embodiments, MALS is performed using a sample cell volume of about 1 μL to 50 μL, e.g., about 2 μL to 25 μL, 5 μL to 20 μL, 1 μL to 40 μL, 1 μL to 30 μL, 1 μL to 20 μL, 1 μL to 10 μL, 40 μL to 50 μL, 30 μL to 50 μL, 20 μL to 50 μL, 10 μL to 50 μL, 5 μL to 15 μL, or 10 μL to 20 μL, e.g., about 10 μL. In some embodiments, MALS is performed using a scattering volume of about 0.001 μL to 0.1 μL, e.g., about 0.005 μL to 0.05 μL, 0.001 μL to 0.05 μL, 0.001 μL to 0.01 μL, 0.05 μL to 0.1 μL, 0.01 μL to 0.1 μL, 0.005 μL to 0.1 μL, or 0.005 μL to 0.05 μL, e.g., about 0.01 μL. In some embodiments, MALS is performed at a temperature range of about 20°C to 70°C, e.g., about 25°C to 75°C or 30°C to 60°C. In some embodiments, MALS is performed with a temperature stability of no more than ±1°C, e.g., no more than ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, or ±0.1°C. In some embodiments, MALS is performed at a pH range of about 1-12, e.g., about 2-11, 2-10, 3-10, 4-9, 5-8, or 6-7.

[0144] In some embodiments, the size of the LNP is determined, for example, the hydrodynamic radius (R h In some embodiments, the molecular weight (MW) of the LNP is determined, for example, the weight average MW (MW) w ), number average MW(M n ), MW corresponding to the maximum value of the chromatographic peak (M p ), z-mean MW(M z or M z+1 ) or viscosity average MW (M v ) in one or more (e.g., 2, 3, 4, or 5). In some embodiments, the polydispersity index is determined, for example, by calculating M w With M nIn some embodiments, determining the R of LNP gw , for example, using Zimm or partial Zimm methods (e.g., as described in Wyatt.Analytica ChimicaActa.1993.272:1-40). In some embodiments, the half-life of the LNP is determined. In some embodiments, the stability of the LNP is determined according to the methods described herein, for example, the methods described in Example 1.

[0145] In some embodiments, the MALS signals obtained in any of the methods described herein are compared directly. In some embodiments, the MALS signals obtained in any of the methods described herein are compared indirectly.

[0146] Lipid Nanoparticles (LNP)

[0147] The systems and methods described herein can be used to analyze lipid nanoparticles.

[0148] Lipid nanoparticle (LNP) is the nanoparticle that is made up of lipid.LNP is generally spherical, and average diameter is between 10 to 1000 nanometers.Solid lipid nanoparticle has solid lipid core matrix, and it can dissolve lipophilic molecules.Lipid core is stabilized by surfactant (emulsifier).The emulsifier used depends on route of administration and has more restrictions for parenteral administration.Term lipid is used in this article with wider meaning and includes triglyceride (for example tristearin), diglyceride (for example behenic acid glyceride), monoglyceride (for example monostearin), fatty acid (for example stearic acid), steroid (for example cholesterol) and wax (for example palmitic acid cetyl ester).All kinds of emulsifiers (in terms of charge and molecular weight) can be used for stabilizing lipid dispersion.In some cases, the combination of emulsifier can prevent particle agglomeration more effectively. For example, LNPs used in some mRNA vaccines can be made from four types of lipids: ionizable cationic lipids (whose positive charge binds to the negatively charged mRNA), PEGylated lipids (for stability), phospholipids (for structure), and cholesterol (for structure).

[0149] In some embodiments, the method does not include one or more (e.g., two or all) of the following steps: a step of recovering LNP, e.g., by ultracentrifugation; a step of diluting the sample, e.g., removing high molecular weight components (e.g., plasma or serum components) that interfere with detection (e.g., detected by DLS); or a step of labeling LNP, e.g., labeling LNP with a fluorophore. In some embodiments, LNP is not recovered, e.g., by ultracentrifugation. In some embodiments, LNP is not diluted, e.g., removing high molecular weight components (e.g., plasma or serum components) that interfere with detection (e.g., detected by DLS). In some embodiments, LNP is not labeled, e.g., LNP is not labeled with a fluorophore.

[0150] In some embodiments, LNP loads nucleic acid. In some embodiments, LNP does not load nucleic acid. In some embodiments, the nucleic acid is a therapeutic nucleic acid (TNA). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a vaccine. In some embodiments, the nucleic acid is a non-coding RNA, for example, a small non-coding RNA. In some embodiments, the nucleic acid is a small interfering RNA (siRNA), an antisense oligonucleotide (ASO) or a micro RNA (miRNA). In some embodiments, the nucleic acid is a guide RNA (gRNA). In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an aptamer. In some embodiments, the nucleic acid includes one or more modified nucleotides. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded.

[0151] In some embodiments, a plurality of LNPs in the analysis sample, for example, according to methods described herein. In some embodiments, at least 50%, 60%, 70%, 80%, 90% or 90% LNP load nucleic acid (for example, nucleic acid described herein) in the sample. In some embodiments, the purity of the LNP in the sample is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In some embodiments, the LNP in the method quantitative detection sample.

[0152] sample

[0153] The methods and systems described herein can be used to analyze a variety of sample types.

[0154] In some embodiments, the method includes obtaining a sample. In some embodiments, the sample comprises a physiological fluid. In some embodiments, the physiological fluid is plasma. In some embodiments, the physiological fluid is serum. In some embodiments, the physiological fluid is blood. In some embodiments, the physiological fluid is amniotic fluid, aqueous humor, bile, breast milk, cerebrospinal fluid, cerumen, chyle, exudate, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural effusion, pus, saliva, sebum, serous fluid, semen, sputum, synovial fluid, sweat, tears, urine, or vomit.

[0155] In some embodiments, the sample or physiological fluid has a pH of 2-10, e.g., 3-9, 4-8, 5-7, 2-8, 2-6, 2-4, 8-10, 6-10, 4-10, 2-10, 2-4, 3-5, 4-6, 6-8, or 7-9. In some embodiments, the sample or physiological fluid has a pH of serum or plasma (e.g., 7.3-7.5). In some embodiments, the sample or physiological fluid has a pH of a tumor microenvironment (e.g., pH 5.6 to 6.8).

[0156] In some embodiments, the sample is diluted after being obtained from the subject. In some embodiments, the sample is not diluted after being obtained from the subject.

[0157] In some embodiments, the sample is not obtained from a subject, e.g., a physiological fluid reconstituted with LNPs. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject is a healthy subject. In some embodiments, the subject suffers from or may suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder. In some embodiments, the subject is a human. In some embodiments, the subject is an animal (e.g., a non-human animal), e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0158] Enumeration implementation

[0159] 1. A method for analyzing lipid nanoparticles (LNP), comprising:

[0160] (optionally) obtaining a sample comprising LNPs (e.g., in a physiological fluid);

[0161] performing size exclusion chromatography (SEC) on the sample; and

[0162] obtaining a multi-angle light scattering (MALS) signal from the sample,

[0163] The LNPs were thereby analyzed.

[0164] 2. The method according to embodiment 1, further comprising:

[0165] Performing SEC on a reference sample; and

[0166] A MALS signal is obtained from the reference sample.

[0167] 3. The method according to embodiment 2 further comprises comparing the MALS signal from the sample with the MALS signal from the reference sample.

[0168] 4. The method according to any one of the preceding embodiments, further comprising performing LC-MS / MS on the sample, for example, to determine (eg, quantify) the components of the LNP.

[0169] 5. The method according to any one of the preceding embodiments, further comprising obtaining the UV absorbance of the sample (e.g., at λ=260 nm), for example, to determine (e.g., quantify) the amount of nucleic acid in the LNP.

[0170] 6. The method according to any one of the preceding embodiments, wherein the method determines the stability of LNPs in the sample.

[0171] 7. The method according to any of the preceding embodiments, wherein the method determines the purity of LNPs in the sample.

[0172] 8. The method according to any one of the preceding embodiments, wherein the method is suitable for monitoring the preparation of the LNP.

[0173] 9. The method according to any one of the preceding embodiments, wherein the method is suitable for determining the pharmacokinetics (PK) of the LNP.

[0174] 10. A method according to any one of embodiments 1-9, wherein the sample is not obtained from a subject, for example, a physiological fluid reconstituted with the LNP.

[0175] 11. A method according to any one of embodiments 1-9, wherein the sample is obtained from a subject.

[0176] 12. The method according to embodiment 10 or 11, wherein the subject is a healthy subject.

[0177] 13. The method of embodiment 10 or 11, wherein the subject suffers from or is likely to suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder.

[0178] 14. The method of any one of embodiments 10-13, wherein the subject is a human.

[0179] 15. A method according to any one of embodiments 10-13, wherein the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0180] 16. A method for determining the stability of a lipid nanoparticle (LNP) in a subject, comprising:

[0181] (optionally) obtaining a first sample comprising LNPs from the subject;

[0182] performing size exclusion chromatography (SEC) on the first sample;

[0183] obtaining a multi-angle light scattering (MALS) signal from the first sample;

[0184] (optionally) obtaining a second sample comprising LNPs from the subject;

[0185] performing SEC on the second sample;

[0186] obtaining a second MALS signal from the second sample; and

[0187] comparing the first MALS signal to the second MALS signal,

[0188] wherein a comparison between the first MALS signal and the second MALS signal is indicative of stability of the LNP in the subject,

[0189] The stability of the LNP in the subject is thereby determined.

[0190] 17. A method according to embodiment 16, wherein the subject has been administered the LNP, for example, at least 1, 6, 12, 18 or 24 hours or 1, 2, 3, 4, 5, 6 or 7 days before obtaining the first sample comprising the LNP.

[0191] 18. A method according to embodiment 16 or 17, wherein the second sample is obtained after the first sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the first sample is obtained.

[0192] 19. The method according to any one of embodiments 16-18, further comprising:

[0193] (optionally) obtaining a third sample comprising the LNP from the subject;

[0194] performing SEC on the third sample;

[0195] obtaining a third MALS signal from the third sample; and

[0196] comparing the third MALS signal to the first MALS signal, the second MALS signal, or both,

[0197] wherein the comparison is indicative of the stability of the LNP in the subject.

[0198] 20. A method according to embodiment 19, wherein the third sample is obtained after the second sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the second sample is obtained.

[0199] 21. The method according to embodiment 19 or 20, further comprising:

[0200] (optionally) obtaining a fourth sample comprising the LNP from the subject;

[0201] performing SEC on the fourth sample;

[0202] obtaining a fourth MALS signal from the fourth sample; and

[0203] comparing the fourth MALS signal to one or more (e.g., all) of the first MALS signal, the second MALS signal, or the third MALS signal,

[0204] wherein the comparison is indicative of the stability of the LNP in the subject.

[0205] 22. A method according to embodiment 21, wherein the fourth sample is obtained after the third sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the third sample is obtained.

[0206] 23. The method according to embodiment 21 or 22, further comprising:

[0207] (optionally) obtaining a fifth sample comprising the LNP from the subject;

[0208] performing SEC on the fifth sample;

[0209] obtaining a fifth MALS signal from the fifth sample; and

[0210] comparing the fifth MALS signal to one or more (e.g., all) of the first MALS signal, the second MALS signal, the third MALS signal, or the fourth MALS signal,

[0211] wherein the comparison is indicative of the stability of the LNP in the subject.

[0212] 24. A method according to embodiment 23, wherein the fifth sample is obtained after the fourth sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the fourth sample is obtained.

[0213] 25. The method of any one of embodiments 16-24, wherein the LNPs in the second or subsequent sample have a change in LNP composition, e.g., lipid, nucleic acid, or both, compared to the LNPs in the first or previous sample, optionally wherein the size of the LNPs in the second or subsequent sample (e.g., R h ) and the size of the LNPs in the first or previous sample (e.g., R h ) are basically the same.

[0214] 26. A method according to any one of embodiments 16-25, wherein the subject is a healthy subject.

[0215] 27. The method of any one of embodiments 16-25, wherein the subject has or is likely to have a disease, e.g., an infection, cancer, or an autoimmune disorder.

[0216] 28. The method of any one of embodiments 16-27, wherein the subject is human.

[0217] 29. A method according to any one of embodiments 16-27, wherein the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0218] 30. A method for determining the stability of lipid nanoparticles (LNPs) in a sample, comprising:

[0219] (optionally) obtaining a first aliquot of the sample;

[0220] subjecting the first aliquot to size exclusion chromatography (SEC);

[0221] obtaining a multi-angle light scattering (MALS) signal from the first aliquot;

[0222] (optionally) obtaining a second aliquot of the sample;

[0223] performing SEC on the second aliquot;

[0224] obtaining a second MALS signal from the second aliquot; and

[0225] comparing the first MALS signal to the second MALS signal,

[0226] wherein a comparison between the first MALS signal and the second MALS signal indicates the stability of the LNPs in the sample,

[0227] The stability of the LNPs in the samples was thereby determined.

[0228] 31. A method according to embodiment 30, wherein the second aliquot is obtained after obtaining the first sample, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after obtaining the first aliquot.

[0229] 32. The method according to embodiment 30 or 31, further comprising:

[0230] (optionally) obtaining a third aliquot of the sample;

[0231] performing SEC on the third aliquot;

[0232] obtaining a third MALS signal from the third aliquot;

[0233] comparing the third MALS signal to the first MALS signal, the second MALS signal, or both,

[0234] wherein the comparing is indicative of the stability of the LNPs in the sample.

[0235] 33. A method according to embodiment 32, wherein the third aliquot is obtained after the second aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the second aliquot is obtained.

[0236] 34. The method according to embodiment 32 or 33, further comprising:

[0237] (optionally) obtaining a fourth aliquot of the sample;

[0238] performing SEC on the fourth aliquot;

[0239] obtaining a fourth MALS signal from the fourth aliquot; and

[0240] comparing the fourth MALS signal to one or more (e.g., all) of the first MALS signal, the second MALS signal, or the third MALS signal,

[0241] wherein the comparing is indicative of the stability of the LNPs in the sample.

[0242] 35. A method according to embodiment 34, wherein the fourth aliquot is obtained after the third aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the third aliquot is obtained.

[0243] 36. The method according to embodiment 34 or 35, further comprising:

[0244] (optionally) obtaining a fifth aliquot of the sample;

[0245] performing SEC on the fifth aliquot;

[0246] obtaining a fifth MALS signal from the fifth aliquot;

[0247] comparing the fifth MALS signal to one or more (e.g., all) of the first MALS signal, the second MALS signal, the third MALS signal, or the fourth MALS signal,

[0248] wherein the comparing is indicative of the stability of the LNPs in the sample.

[0249] 37. A method according to embodiment 36, wherein the fifth aliquot is obtained after the fourth aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the fourth aliquot is obtained.

[0250] 38. The method of any one of embodiments 30-37, wherein the LNPs in the second or subsequent sample have a change in LNP composition, e.g., lipid, nucleic acid, or both, compared to the LNPs in the first or previous sample, optionally wherein the size (e.g., R h ) and the size of the LNPs in the first or previous sample (e.g., R h ) are basically the same.

[0251] 39. A method according to any one of embodiments 30-38, wherein the sample is not obtained from a subject, e.g., a physiological fluid reconstituted with the LNP.

[0252] 40. A method according to any one of embodiments 30-38, wherein the sample is obtained from a subject.

[0253] 41. The method of embodiment 39 or 40, wherein the subject is a healthy subject.

[0254] 42. The method of embodiment 39 or 40, wherein the subject suffers from or is likely to suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder.

[0255] 43. A method according to any one of embodiments 39-42, wherein the subject is human.

[0256] 44. A method according to any one of embodiments 39-42, wherein the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0257] 45. A method for determining the purity of lipid nanoparticles (LNPs) in a sample, comprising:

[0258] (optionally) obtaining a first aliquot of the sample;

[0259] subjecting the first aliquot to size exclusion chromatography (SEC);

[0260] obtaining a multi-angle light scattering (MALS) signal from the first aliquot;

[0261] (optionally) obtaining a second aliquot of the sample;

[0262] performing SEC on the second aliquot;

[0263] obtaining a second MALS signal from the second aliquot; and

[0264] comparing the first MALS signal to the second MALS signal to determine the stability of the LNPs in the sample, which is indicative of the purity of the LNPs in the sample,

[0265] The purity of LNPs in the sample was thereby determined.

[0266] 46. ​​A method according to embodiment 45, wherein the second aliquot is obtained after the first aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the first aliquot is obtained.

[0267] 47. The method according to embodiment 45 or 46, further comprising:

[0268] (optionally) obtaining a first reference aliquot of a reference sample comprising LNPs;

[0269] performing SEC on the first reference aliquot;

[0270] obtaining a MALS signal from the first reference aliquot;

[0271] (optionally) obtaining a second reference aliquot of the reference sample;

[0272] performing SEC on the second reference aliquot;

[0273] obtaining a second MALS signal from the second reference aliquot;

[0274] The first MALS signal is compared to the second MALS signal to determine the stability of the LNPs in the reference sample.

[0275] 48. The method of any one of embodiments 45-47, further comprising comparing the stability of the LNP in the sample with the stability of the LNP in the reference sample to determine the purity of the LNP in the sample.

[0276] 49. The method according to any one of embodiments 45-48, further comprising:

[0277] (optionally) obtaining a third aliquot of the sample (or the reference sample);

[0278] performing SEC on the third aliquot;

[0279] obtaining a third MALS signal from the third aliquot;

[0280] The third MALS signal is compared to the first MALS signal, the second MALS signal, or both.

[0281] 50. A method according to embodiment 49, wherein the third aliquot is obtained after the second aliquot is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the second aliquot is obtained.

[0282] 51. The method according to embodiment 49 or 50, further comprising:

[0283] (optionally) obtaining a fourth aliquot of the sample (or the reference sample);

[0284] performing SEC on the fourth aliquot;

[0285] obtaining a fourth MALS signal from the fourth aliquot; and

[0286] The fourth MALS signal is compared to one or more (eg, all) of the first MALS signal, the second MALS signal, or the third MALS signal.

[0287] 52. A method according to embodiment 51, wherein the fourth aliquot is obtained after the third sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the third aliquot is obtained.

[0288] 53. The method according to embodiment 51 or 52, further comprising:

[0289] (optionally) obtaining a fifth aliquot of the sample (or the reference sample);

[0290] performing SEC on the fifth aliquot;

[0291] obtaining a fifth MALS signal from the fifth aliquot;

[0292] The fifth MALS signal is compared to one or more (eg, all) of the first MALS signal, the second MALS signal, the third MALS signal, or the fourth MALS signal.

[0293] 54. A method according to embodiment 53, wherein the fifth aliquot is obtained after the fourth sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the fourth aliquot is obtained.

[0294] 55. A method according to any one of embodiments 45-54, wherein the LNPs in the sample have impurities due to an incompletely deprotected form of the LNPs.

[0295] 56. A method according to any one of embodiments 45-55, wherein the sample is not obtained from a subject, e.g., a physiological fluid reconstituted with the LNP.

[0296] 57. A method according to any one of embodiments 45-55, wherein the sample is obtained from a subject.

[0297] 58. A method according to embodiment 56 or 57, wherein the subject is a healthy subject.

[0298] 59. The method of embodiment 56 or 57, wherein the subject suffers from or is likely to suffer from a disease, e.g., an infection, cancer, or an autoimmune disorder.

[0299] 60. The method of any one of embodiments 56-59, wherein the subject is human.

[0300] 61. A method according to any one of embodiments 56-59, wherein the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0301] 62. A method for monitoring the preparation process of lipid nanoparticles (LNP), comprising:

[0302] (optionally) obtaining a first sample comprising the LNP from a process for preparing the LNP;

[0303] performing size exclusion chromatography (SEC) on the first sample; and

[0304] obtaining a multi-angle light scattering (MALS) signal from the first sample,

[0305] Thereby monitoring the preparation process of the LNP.

[0306] 63. The method according to embodiment 62, further comprising:

[0307] (optionally) obtaining a second sample comprising the LNP from the process of preparing the LNP;

[0308] performing SEC on the second sample; and

[0309] A second MALS signal is obtained from the second sample.

[0310] 64. A method according to embodiment 63, wherein the second sample is obtained after the first sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the first sample is obtained.

[0311] 65. The method according to embodiment 63 or 64, further comprising:

[0312] (optionally) obtaining a third sample comprising the LNP from the process of preparing the LNP;

[0313] performing SEC on the third sample; and

[0314] A third MALS signal is obtained from the third sample.

[0315] 66. A method according to embodiment 65, wherein the third sample is obtained after the second sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the second sample is obtained.

[0316] 67. The method according to embodiment 65 or 66, further comprising:

[0317] (optionally) obtaining a fourth sample comprising the LNP from the process of preparing the LNP;

[0318] performing SEC on the fourth sample; and

[0319] A fourth MALS signal is obtained from the fourth sample.

[0320] 68. A method according to embodiment 67, wherein the fourth sample is obtained after the third sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the third sample is obtained.

[0321] 69. The method according to embodiment 67 or 68, further comprising:

[0322] (optionally) obtaining a fifth sample comprising the LNP from the process of preparing the LNP;

[0323] performing SEC on the fifth sample; and

[0324] A fifth MALS signal is obtained from the fifth sample.

[0325] 70. A method according to embodiment 69, wherein the fifth sample is obtained after the fourth sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the fourth sample is obtained.

[0326] 71. A method for determining the pharmacokinetics (PK) of a lipid nanoparticle (LNP), comprising:

[0327] (optionally) obtaining a first sample comprising LNPs from a subject;

[0328] performing size exclusion chromatography (SEC) on the first sample; and

[0329] obtaining a multi-angle light scattering (MALS) signal from the first sample,

[0330] The PK of the LNP is thereby determined.

[0331] 72. The method according to embodiment 71, further comprising:

[0332] (optionally) obtaining a second sample comprising the LNP from the subject;

[0333] performing SEC on the second sample; and

[0334] A second MALS signal is obtained from the second sample.

[0335] 73. A method according to embodiment 72, wherein the second sample is obtained after the first sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the first sample is obtained.

[0336] 74. The method according to embodiment 72 or 73, further comprising:

[0337] (optionally) obtaining a third sample comprising the LNP from the subject;

[0338] performing SEC on the third sample; and

[0339] A third MALS signal is obtained from the third sample.

[0340] 75. A method according to embodiment 74, wherein the third sample is obtained after the second sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the second sample is obtained.

[0341] 76. The method according to embodiment 73 or 74, further comprising:

[0342] (optionally) obtaining a fourth sample comprising the LNP from the subject;

[0343] performing SEC on the fourth sample; and

[0344] A fourth MALS signal is obtained from the fourth sample.

[0345] 77. A method according to embodiment 76, wherein the fourth sample is obtained after the third sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the third sample is obtained.

[0346] 78. The method according to embodiment 76 or 77, further comprising:

[0347] (optionally) obtaining a fifth sample comprising the LNP from the subject;

[0348] performing SEC on the fifth sample; and

[0349] A fifth MALS signal is obtained from the fifth sample.

[0350] 79. A method according to embodiment 78, wherein the fifth sample is obtained after the fourth sample is obtained, for example, at least 15, 30, 45, 60, 75, 90, 105 or 120 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36 or 48 hours after the fourth sample is obtained.

[0351] 80. A method according to any one of embodiments 71-79, wherein the subject is a healthy subject.

[0352] 81. The method of any one of embodiments 71-79, wherein the subject has or is likely to have a disease, e.g., an infection, cancer, or an autoimmune disorder.

[0353] 82. The method of any one of embodiments 71-81, wherein the subject is human.

[0354] 83. The method of any one of embodiments 71-81, wherein the subject is an animal, e.g., a mammal, e.g., a mouse, a rat, or a primate.

[0355] 84. The method of any one of embodiments 1-83, wherein the SEC is performed in a dual column configuration.

[0356] 85. A method according to any one of embodiments 1-83, wherein the SEC is performed in a dual column configuration, for example, using two columns with different pore sizes, for example, a first column with a first average pore size and a second column with a second average pore size.

[0357] 86. A method according to embodiment 85, wherein the first average pore size is greater than the second average pore size.

[0358] 87. A method according to embodiment 85 or 86, wherein the first average pore size has a molecular weight cutoff (MWCO) of about 1 MDa or greater, for example, about 2 MDa or greater, 5 MDa or greater, 10 MDa or greater, 15 MDa or greater or 20 MDa or greater.

[0359] 88. A method according to any of embodiments 85-87, wherein the first average pore size has a MWCO of about 20 MDa or less, for example, about 15 MDa or less, 10 MDa or less, 5 MDa or less, 2 MDa or less, or 1 MDa or less.

[0360] 89. The method of any one of embodiments 85-88, wherein the first average pore size has a MWCO of about 1 MDa to 20 MDa, for example, about 2 MDa to 15 MDa, 5 MDa to 10 MDa, 1 MDa to 15 MDa, 1 MDa to 10 MDa, 1 MDa to 5 MDa, 1 MDa to 2 MDa, 15 MDa to 20 MDa, 10 MDa to 20 MDa, 5 MDa to 20 MDa, 2 MDa to 20 MDa, 2 MDa to 10 MDa, 5 MDa to 15 MDa, or 8 MDa to 12 MDa, for example, about 10 MDa.

[0361] 90. A method according to any of embodiments 85-89, wherein the second average pore size has a MWCO of about 10 MDa or less, for example, about 5 MDa or less, 2 MDa or less, 1 MDa or less, 0.5 MDa or less, 0.2 MDa or less or 0.1 MDa or less.

[0362] 91. A method according to any of embodiments 85-90, wherein the second average pore size has a MWCO of about 0.1 MDa or greater, for example, about 0.2 MDa or greater, 0.5 MDa or greater, 1 MDa or greater, 2 MDa or greater, 5 MDa or greater, or 10 MDa or greater.

[0363] 92. A method according to any one of embodiments 85-91, wherein the second average pore size has a MWCO of about 0.1 MDa to 10 MDa, for example, about 0.2 MDa to 5 MDa, 0.5 MDa to 2 MDa, 0.1 MDa to 5 MDa, 0.1 MDa to 2 MDa, 0.1 MDa to 1 MDa, 0.1 MDa to 0.5 MDa, 0.1 MDa to 0.2 MDa, 5 MDa to 10 MDa, 2 MDa to 10 MDa, 1 MDa to 10 MDa, 0.5 MDa to 10 MDa, 0.2 MDa to 10 MDa, 0.2 MDa to 1 MDa, 1 MDa to 5 MDa or 0.3 MDa to 0.7 MDa, for example, about 0.5 MDa.

[0364] 93. A method according to any one of embodiments 1-92, wherein the SEC is performed using a column having a length of 100 mm or longer, for example, about 150 mm or longer, 200 mm or longer, 250 mm or longer, 300 mm or longer, 400 mm or longer, 500 mm or longer, 600 mm or longer, 700 mm or longer, 800 mm or longer, 900 mm or longer, or 1000 mm or longer, for example, about 100 mm to 1000 mm, 150 mm to 500 mm, or about 200 mm to 300 mm, for example, about 250 mm.

[0365] 94. The method of any one of embodiments 1-93, wherein the SEC is performed using a polymer matrix column.

[0366] 95. The method of any one of embodiments 1-93, wherein the SEC is not performed using a silica-based column.

[0367] 96. A method according to any one of embodiments 1-95, wherein the SEC is performed under aqueous conditions, for example, using a biocompatible buffer system, for example, phosphate buffer, bicarbonate buffer, acetate buffer, Tris buffer, or Good's buffer (e.g., as described in Good et al. Biochemistry. 1966; 5(2):467-77; Good and Izawa Methods Enzymol. 1972; 24:53-68; Ferguson et al. Anal. Biochem. 1980 May; 104(2): 300-10), for example, any one of MES, Bis-tris methane, ADA, Bis-tris propane, PIPES, ACES, MOPSO, choline chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetylaminoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tris(hydroxymethyl)methylglycine (Tricine), tris(hydroxymethyl)aminomethane (Tris), glycinamide, glycine, HEPBS, Bicine, TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS or any one of a combination thereof.

[0368] 97. The method of any one of embodiments 1-96, wherein the SEC is performed without using an organic solvent (eg, THF) as the mobile phase.

[0369] 98. A method according to any one of embodiments 1-97, wherein the MALS is performed at scattering angles of 15° and 90°.

[0370] 99. The method of any one of embodiments 1-98, wherein the MALS is performed using a laser wavelength of about 500 nm to 800 nm, for example, about 600 nm to 700 nm, 500 nm to 700 nm, 600 nm to 800 nm, 500 nm to 600 nm, 700 nm to 800 nm, for example, about 658 nm.

[0371] 100. A method according to any one of embodiments 1-99, wherein the MALS is performed using a sample pool volume of about 1 μL to 50 μL, for example, about 2 μL to 25 μL, 5 μL to 20 μL, 1 μL to 40 μL, 1 μL to 30 μL, 1 μL to 20 μL, 1 μL to 10 μL, 40 μL to 50 μL, 30 μL to 50 μL, 20 μL to 50 μL, 10 μL to 50 μL, 5 μL to 15 μL or 10 μL to 20 μL, for example, about 10 μL.

[0372] 101. A method according to any one of embodiments 1-100, wherein the MALS is performed using a scattering volume of about 0.001 μL to 0.1 μL, for example, about 0.005 μL to 0.05 μL, 0.001 μL to 0.05 μL, 0.001 μL to 0.01 μL, 0.05 μL to 0.1 μL, 0.01 μL to 0.1 μL, 0.005 μL to 0.1 μL or 0.005 μL to 0.05 μL, for example, about 0.01 μL.

[0373] 102. The method of any one of embodiments 1-101, wherein the MALS is performed at a temperature range of about 20°C to 70°C, for example, about 25°C to 75°C or 30°C to 60°C.

[0374] 103. The method of any one of embodiments 1-102, wherein the MALS is performed with a temperature stability of no more than ±1°C, e.g., no more than ±0.5°C or ±0.2°C.

[0375] 104. The method of any one of embodiments 1-103, wherein the MALS is performed at a pH range of about 1-12, e.g., about 2-11 or 2-10.

[0376] 105. The method of any one of embodiments 1-104, wherein the size of the LNP is determined, for example, the hydrodynamic radius (R h ).

[0377] 106. The method of any one of embodiments 1-105, wherein the molecular weight (MW) of the LNP is determined, for example, the weight average MW (MW) is determined. w ), number average MW(M n ), MW corresponding to the maximum value of the chromatographic peak (M p ), z-mean MW(M z or M z+1 ) or viscosity average MW (M v ) in one or more (e.g., 2, 3, 4 or 5).

[0378] 107. The method of any one of embodiments 1-106, wherein the polydispersity index is determined, for example, by calculating M w With M n ratio.

[0379] 108. The method of any one of embodiments 1-107, wherein the R of the LNP is determined gw , for example, using Zimm or partial Zimm methods.

[0380] 109. The method of any one of embodiments 1-108, wherein the half-life of the LNP is determined.

[0381] 110. The method of any one of embodiments 1-109, comprising directly or indirectly comparing the obtained MALS signals.

[0382] 111. The method of any one of embodiments 1-110, wherein multiple LNPs in the sample are analyzed.

[0383] 112. The method of any one of embodiments 1-111, wherein the method does not comprise a step of recovering the LNP, e.g., by ultracentrifugation.

[0384] 113. A method according to any one of embodiments 1-112, wherein the method does not include a step of diluting the sample, for example, to remove high molecular weight components (e.g., plasma or serum components) that interfere with detection (e.g., detection by DLS).

[0385] 114. A method according to any one of embodiments 1-113, wherein the method does not include a step of labeling the LNP, for example, labeling the LNP with a fluorophore.

[0386] 115. The method of any one of embodiments 1-114, wherein the method quantitatively detects LNPs in the sample.

[0387] 116. A method according to any one of embodiments 1-115, wherein the LNP is loaded with nucleic acid.

[0388] 117. A method according to any one of embodiments 1-115, wherein the LNP is not loaded with nucleic acid.

[0389] 118. The method of embodiment 116 or 117, wherein the nucleic acid is a therapeutic nucleic acid (TNA).

[0390] 119. The method of any one of embodiments 116-118, wherein the nucleic acid is mRNA.

[0391] 120. The method of any one of embodiments 116-119, wherein the nucleic acid is a vaccine.

[0392] 121. The method of any one of embodiments 116-120, wherein the nucleic acid is a non-coding RNA, e.g., a small non-coding RNA.

[0393] 122. The method of any one of embodiments 116-121, wherein the nucleic acid is a small interfering RNA (siRNA), an antisense oligonucleotide (ASO), or a micro RNA (miRNA).

[0394] 123. The method of any one of embodiments 116-118, wherein the nucleic acid is DNA.

[0395] 124. The method of any one of embodiments 116-118, wherein the nucleic acid is an aptamer.

[0396] 125. The method of any one of embodiments 116-124, wherein the nucleic acid comprises one or more modified nucleotides.

[0397] 126. A method according to any one of embodiments 116-125, wherein the nucleic acid is single-stranded.

[0398] 127. The method of any one of embodiments 116-125, wherein the nucleic acid is double-stranded.

[0399] 128. The method of any one of embodiments 1-127, wherein the sample comprises a physiological fluid.

[0400] 129. A method according to embodiment 128, wherein the physiological fluid is plasma.

[0401] 130. The method of embodiment 128, wherein the physiological fluid is serum.

[0402] 131. A method according to embodiment 128, wherein the physiological fluid is blood.

[0403] 132. The method of embodiment 128, wherein the physiological fluid is amniotic fluid, aqueous humor, bile, breast milk, cerebrospinal fluid, cerumen, chyle, exudate, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural effusion, pus, saliva, sebum, serum, semen, sputum, synovial fluid, sweat, tears, urine or vomit.

[0404] 133. The method of any one of embodiments 128-132, wherein the physiological fluid has a pH of 2-10, e.g., 3-9, 4-8, 5-7, 2-8, 2-6, 2-4, 8-10, 6-10, 4-10, 2-10, 2-4, 3-5, 4-6, 6-8, or 7-9.

[0405] 134. The method of embodiment 133, wherein the physiological fluid has a pH of serum or plasma (e.g., 7.3-7.5).

[0406] 135. A method according to embodiment 133, wherein the physiological fluid has a pH of a tumor microenvironment (e.g., pH 5.6 to 6.8).

[0407] 136. A system comprising a processor configured to perform the method according to any one of embodiments 1-35.

[0408] 137. A system according to embodiment 136, wherein the processor is configured to perform size exclusion chromatography (SEC) on the sample or an aliquot of the sample.

[0409] 138. A system according to embodiment 136 or 137, wherein the processor is also configured to obtain a multi-angle light scattering signal (MALS) from the sample or an aliquot of the sample.

[0410] Example

[0411] Example 1. Quantitative analysis of lipid nanoparticle stability in physiological fluids by size exclusion chromatography combined with multi-angle light scattering

[0412] Introduction

[0413] This example demonstrates that the stability of LNPs in undiluted plasma and serum can be accurately measured using size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). An exemplary workflow for measuring LNP stability is shown in FIG. Figure 1A As shown in Figure 2, LNPs degraded with first-order kinetics in both plasma and serum, with a half-life of approximately 85 min at room temperature. The apparent Mw and R g The changes in indicated that LNP degradation in plasma was associated with replacement of amino-, PEG- and auxiliary lipids by proteins over time. In contrast, LNP composition was not affected by serum. Finally, in this study, LNP stability in plasma was affected by trace impurities (about 1%) in the lipid composition. In summary, these findings indicate that SEC-MALS is a useful tool for lipid formulation optimization and quality control.

[0414] method

[0415] Human plasma powder (P9523-5ML), DDC Mass Spect Gold serum (MSG3000-100ML), and human serum albumin (A3782-5G) were purchased from Sigma Aldrich.

[0416] Fabrication of mRNA-LNPs – Lipid nanoparticle components ALC-0315, ALC-0159, DSPC, and cholesterol were purchased from MedhemExpress (HY-138170, HY-138300, HY-W040193, and HY-N0322, respectively). mRNA was in vitro transcribed from a 3800 nt PCR-amplified dsDNA template using T7 RNA polymerase (NEB, M0251L), purified Monarch RNA Cleanup Kit spin columns (NEB T2040L), quantified by UV spectroscopy, and then dissolved in 1 mM sodium acetate buffer (pH 4.8) to form an aqueous phase. mRNA-LNPs were fabricated using the same composition as Cominarty. ALC-0315, ALC-0159, DSPC, and cholesterol were dissolved in ethanol at a molar ratio of 46.3:1.6:9.4:42.7 to form an organic phase. The organic phase and aqueous phase with a volume ratio of 3: 1 were mixed at a flow rate of 12 mL / min in a benchtop microfluidic device (NanoAssemblr Platform, Precision NanoSystems). The N / P ratio for preparing mRNA-LNP was 6: 1. The resulting mRNA-LNP buffer was exchanged into a saline solution and concentrated to a total lipid concentration of ~2.2 mg / mL, and then characterized by batch DLS (Zetasizer, Malvern Panalytical) or SEC-MALS. Empty LNPs were prepared in the same way, but without mRNA.

[0417] Incubation of mRNA-LNPs in human plasma, serum, and albumin – Human plasma powder was reconstituted in MilliQ water according to the manufacturer’s instructions, then centrifuged at 10,000 x g for 10 min and filtered through 0.2 μm to remove particulates. Serum albumin was dissolved in 50 mM sodium phosphate buffer (pH 7.2) at a concentration of 40 mg / ml. mRNA-LNPs were diluted 1:10 (v / v) into filtered plasma, serum, or serum albumin solutions and then injected every 15 min for SEC-MALS analysis.

[0418] SEC-MALS – The following equipment stack was used: (i) Agilent 1290 Infinity II Bio LC with quaternary pump, (ii) Agilent 1260 Infinity II Bio-SEC multi-detector system, (iii) Agilent 1290 Infinity II diode array detector, and (iv) Agilent 1290 Infinity II refractive index detector. The diode array detector was mainly used for system calibration and was not required for light scattering experiments, as the refractive index detector was used for concentration estimation. The quaternary pump was used in isocratic mode using only a single solvent line to effectively reduce baseline fluctuations of the refractive index detector. The multi-detector system was configured to enable multi-angle light scattering at 15° and 90° as well as dynamic light scattering. Two 4.6x250mm, 8μm, PL Aquagel-OH SEC columns (PL1549-5801 and PL1549-5800) with different pore sizes were used in this study. The mobile phase was 50 mM sodium phosphate (pH 7.2) filtered three times through 0.1 μm and used at a flow rate of 0.6 ml / min.

[0419] Prior to sample analysis, the multi-detector system and refractive index detector were equilibrated to 30 °C for 24 hours. The SEC column was flushed overnight at a low flow rate directly to waste, bypassing all detectors. Approximately 1 hour before sample analysis, with the flow still bypassing all detectors, the flow rate was gradually increased to 0.6 ml / min. The SEC column was then flushed at 0.6 ml / min for at least 5 min before connecting the SEC column to the detector. The detector was then flushed at 0.6 ml / min until all readings reached a low and stable baseline. Several 50 μl injections of 10 mg / ml BSA dissolved in the mobile phase were made to ensure system suitability and calibrate the system. Data acquisition and analysis were performed in Agilent Bio-SEC software, and for mRNA-LNPs used The value is 0.16, for BSA The value is 0.185.

[0420] LC-MS / MS Quantification of mRNA-LNP Lipid Fractions – This assay was performed on an Agilent 6545XT LC / Q-TOF (front end Agilent 1290 Infinity II Bio LC with binary pump).

[0421] For chromatography, a 2.1 x 50 mm, 1.9 μm InfinityLab Poroshell 120 phenylhexyl column was used. The column was heated to 45 °C for analysis and the flow rate was set at 0.4 ml / min. Mobile phase A was 87% MeOH / 10 mM ammonium acetate and mobile phase B was 90% ACN / 10 mM ammonium acetate. All solvents used were LC-MS grade. Prior to analysis, the column was first equilibrated at 0% mp B. During analysis, the gradient was set to 0% mp B for the first 0-2 minutes, after which mp B was linearly increased to 100% from 2–7 min, followed by a 3 min post time.

[0422] To generate the calibration curve, a stock solution containing 2 mM ALC-0315, 2 mM ALC-0159, 2 mM DSPC and 20 mM cholesterol was freshly prepared in methanol for each analysis. The calibration solution was then serially diluted in methanol to a minimum concentration of 0.1 nM ALC-0315, 0.1 nM ALC-0159, 0.1 nM DSPC and 1 nM cholesterol. Prior to injection, several injections of the diluted calibration solution were performed to ensure consistent separation and mass spectrometry signals.

[0423] For LC-MS profiling of mRNA-LNP fractions, the same chromatography method and mass spectrometer parameters were used, but the mass spectrometer was set to acquire MS1 only.

[0424] Confirm the identity of the ALC-0315 impurity - The impurity fractions after reverse phase chromatography were collected and combined in 2 ml snap cap tubes and then evaporated to dryness in a centrifugal evaporator. The impurity sample was deprotected with 33% HBr / AcOH (0.5 ml) and trifluoroacetic acid (TFA, 2 ml) for 1 hour and then stored refrigerated at 4°C before analysis. The control impurity sample was reconstituted in 87% MeOH for analysis without deprotection.

[0425] result

[0426] mRNA-LNPs and empty LNPs (without mRNA) were diluted into 50 mM phosphate buffer (pH 7.2) to a final lipid concentration of 0.22 mg / ml and then analyzed by SEC-MALS to determine their physical characteristics by deconvolution of refractive index and light scattering of chromatograms at 15° and 90° (LS15° and LS90°) ( Figure 1B SEC-MALS and bulk DLS R h The measurements were in good agreement and indicated that both types of nanoparticles were essentially monodisperse (Table 1). Empty LNPs were smaller in size and had a lower apparent M than mRNA-LNPs. w values, which is consistent with their lack of mRNA loading.

[0427] Table 1. Physical properties of mRNA-LNP and empty LNP (N=3)

[0428]

[0429] Separation in SEC is due to transient inclusion of analytes into porous chromatographic resins, however resolution depends on column length and average pore size. When a single column proves insufficient, columns of different average pore sizes (reflected by their molecular weight cutoff or MWCO) can be used to separate analytes with a wider molecular weight range than can be handled by a single column type. mRNA-LNP and human plasma were injected in a single or dual column configuration, respectively ( Figures 2A-2D The single column configuration included a wide-bore 4.6 x 250 mm PL aquagel-OH column (10 MDa MWCO), and the dual column configuration included a wide-bore column followed in series by a second narrower-bore 4.6 x 250 mm aquagel-OH column (0.5 MDa MWCO). The single column configuration was not able to adequately separate plasma components from mRNA-LNPs ( Figure 2A ), while the dual-column configuration provided acceptable resolution for most of the mRNA-LNP peaks ( Figure 2B ). The refractive index signal from mRNA-LNPs is much smaller than that from plasma components, and co-elution can lead to significant errors in the estimation of mRNA-LNP concentrations, resulting in M w Inaccurate calculation ( Figure 2C-2D ). Therefore, good chromatographic separation is a necessary condition for accurate analysis ( Figure 2D ).

[0430] mRNA-LNPs were incubated in human plasma and injected every 15 min for separation and analysis using dual-column SEC-MALS ( Figure 3A). The LS90° chromatogram clearly shows at 6.7 min that the mRNA-LNP peak intensity decreases over time, indicating that the mRNA-LNP is degraded in human plasma. At 7.7 min, the plasma component peak decreases slightly over time due to the tailing of the mRNA-LNP peak. At 8.2 min, the plasma component peak is baseline-separated from the mRNA-LNP and shows no change in its intensity, retention time, or peak shape throughout the experiment, serving as an internal control for the consistency of the separation quality and injection volume. UV absorbance at λ=260 nm can be used to detect the elution of the encapsulated mRNA, despite the lack of a strong UV chromophore in the nanoparticle lipids. Figure 3B-3C Overlaid UV absorbance and refractive index chromatograms are shown for the same injection series. However, the observed signal is confounded by scattering of incident light by the nanoparticles, making uncorrected UV absorbance an inaccurate means of estimating mRNA-LNP concentration ( Figure 3B ). As UV absorbance and refractive index detectors are much less sensitive to nanoparticles than LS90°, they are less suitable for accurately monitoring LNP degradation kinetics. However, the decrease in mRNA-LNP peak intensity over time confirms the LS90° results ( Figure 3C ). DLS readings for all injections were obtained by R h showed consistent separation of the analytes, confirming that the separation occurred primarily via a size exclusion mechanism ( Figure 3D ).

[0431] The degradation kinetics of mRNA-LNP in human plasma and serum were averaged from three independent experiments ( Figure 4A ). The degradation rate is well described by first-order kinetics and is similar in plasma and serum, with a half-life between 80 and 85 min (blue and orange traces). In contrast, in 40 mg / ml human serum albumin, mRNA-LNP is relatively stable because the peak area does not change significantly after 30 min. Both hydrophobic interactions and Coulomb electrostatic interactions between negatively charged mRNA and positively charged amino-lipids stabilize mRNA-LNP. It is assumed that empty LNP degrades faster than mRNA-LNP because negatively charged proteins in serum can interact with positively charged amino lipids, thereby making the structure of LNP unstable. Since empty LNP has a shorter half-life of 53 min in serum (green trace), this has been confirmed, thus indicating that the observed degradation rate represents LNP stability. In addition, the apparent M of degraded mRNA-LNP w The average value was calculated from three independent experiments. Figure 4B ). The apparent M of mRNA-LNP wIt remained constant at ∼43 MDa in serum but decreased significantly over time in plasma, indicating that degradation processes were different in the two environments. h Remains roughly the same, but the apparent M in plasma still appears w , as indicated by the absence of a change in the retention time of the eluted mRNA-LNP peak ( Figure 3A and Figure 3D ).

[0432] Throughout the experiment, mRNA-LNP lipids were gradually replaced by plasma proteins, thus changing the average composition of degraded mRNA-LNPs while maintaining their average R h Since proteins have a higher average refractive index increment than the average refractive index increment of 0.16 for nanoparticle lipids, =0.19, in which case it is assumed that the average mRNA-LNP degraded in plasma Increases over time. From the light scattering equation, we know that the apparent M w and Inversely correlated:

[0433]

[0434] Where R θ is the light scattering intensity at angle θ, P θ is the scattering function, M w is the apparent molecular weight, is the refractive index increment, C is the concentration, and K is a constant.

[0435] R of degraded mRNA-LNP in serum gw remained roughly unchanged, while the R gw It increases over time ( Figure 4C-4D ). This observation indicates that heavier plasma proteins are replacing lipids around the mRNA-LNPs, resulting in an increase in the mean radius of gyration. To confirm that the mRNA-LNP composition changes in human plasma but not in serum, the molar ratio of cholesterol:ALC-0315:DSPC:ALC-0159 was quantified using an LC-MS / MS method.

[0436] For mass spectrometry analysis, three time periods were planned: 0–1.3 min, where flow was directed to waste, 1.3–4 min, where flow was directed to the mass spectrometer with parameters optimized for cholesterol analysis, and 4–7 min, where flow was directed to the mass spectrometer with parameters optimized for lipid analysis. The following parameters were used:

[0437]

[0438]

[0439] Degraded mRNA-LNPs in plasma and serum were collected at 0 and 90 min, and their lipid components were quantified in three independent experiments.

[0440] For a mixture of LNP component standards injected at different amounts, parallel reaction monitoring (PRM) chromatograms were obtained to demonstrate the repeatability of retention time and baseline separation for each component using the 7-min separation method ( Figure 5A ). Briefly, parallel reaction monitoring was performed in the time periods of 1.3–4 min and 4–7 min, with the following precursor masses and collision energies:

[0441] Time period Accurate m / z Z Retention time ΔRT Iso Width CE 1.3min 369.65 1 1.8 1 4m / z 30 4min 790.82 1 4.8 0.8 4m / z 50 4min 766.91 1 5.7 0.4 4m / z 60 4min 1184.10 2 6.1 0.4 4m / z 100

[0442] Control mRNA-LNPs were not exposed to either serum or plasma and showed a cholesterol:ALC-0315:DSPC:ALC-0159 molar ratio of 42.1:49.2:6.9:1.9 ( Figure 5B-5C ), similar to the theoretical molar ratio for LNPs prepared by microfluidics (42.7:46.3:9.4:1.6). At T=0 min, the ratios of ALC-0315, DSPC, and ALC-0159 relative to cholesterol in both serum and plasma decreased slightly (serum=51.7:41.7:5.3:1.2, plasma=49.2:40.8:8.4:1.5). At T=90 min, the LNP composition in serum showed slight changes in the main components cholesterol and ALC-0315, but further reductions in DSPC and ALC-0159 were significant (54.4:40.7:4.5:0.4). However, when incubated with plasma, in addition to the reduction in the ratios of DSPC and ALC-0159, the ratio of ALC-0315 was also significantly reduced (75.4:20.3:4.0:0.4).

[0443] In summary, both light scattering and LC-MS / MS data indicate that plasma incubation leads to significant changes in the composition of mRNA-LNPs during degradation, even though the nanoparticles retain their original R h Therefore, proteins present in plasma but not in serum, such as coagulation factors (e.g., fibrinogen), appear to preferentially replace the amino lipid ALC-0315 in the mRNA-LNPs. Although LC-MS / MS data showed that the LNP composition also changed during serum incubation, this change occurred mainly through the replacement of the minor lipid components DSPC and ALC-0159, thus resulting in the M w and R gw The overall changes are small.

[0444] This SEC-MALS method can be used to evaluate the effect of trace lipid impurities on the stability of mRNA-LNP. Plasma component standards were identified by MS1 spectroscopy (FIGS.9A-9D). Calibration curves were generated for individual standards, such as Figures 10A-10D Total ion chromatograms of lipid components from two batches of mRNA-LNPs prepared using different lots of ALC-0315, but all other components were prepared using the same lot number ( Figure 6A-6B In addition to the four expected standard components, the impurity-containing batch had a fifth elution peak at 6.5 min ( Figure 6B ), the monoisotopic mass is 866.8208Da ( Figure 6C ). Impurity abundance estimation of batch ALC-0315 was performed using LC-UV method. The chromatogram depicts the elution peaks of ALC-0315 with and without significant impurities ( Figures 11A-11C , 12A-12C, Table 2-3). The MS1 spectrum of the impure batch revealed a strong impurity peak at 866.9 m / z ( Figures 11A-11C ), however, was not present in all batches of ALC-0315 ( Figures 12A-12C ).

[0445] Table 2. Peak list of ALC-0315 sample containing impurities

[0446] peak# Retention time area high area% 1 15.478 25473440 519440 98.907 2 18.776 52502 12086 0.204 3 19.859 8761 2651 0.034 4 20.425 22537 8042 0.088 5 20.828 59517 3014 0.231 6 22.817 138142 16678 0.536 total 25754898 561911 100.00

[0447] Table 3. Peak list of ALC-0315 sample without impurities

[0448] peak# Retention time area high area% 1 14.959 42904743 648135 99.910 2 18.203 8949 79 0.021 3 18.646 9038 2329 0.021 4 20.571 11060 267 0.026 5 22.833 9434 1577 0.022 total 42943225 652387 100.00

[0449] Although the impure mRNA-LNPs had similar physical characteristics to the pure mRNA-LNPs as assessed by bulk DLS (Table 4), they exhibited greatly reduced stability in serum. The impure mRNA-LNPs degraded more rapidly in serum, resulting in an order of magnitude lower abundance (~5%) than the pure mRNA-LNPs (~50%) after 90 min (Table 4). Fig.6D ).

[0450] Table 4. Physical properties of mRNA-LNPs with and without impurities (N=3)

[0451] mRNA-LNP Rh(nm) PD ζ(mV) No impurities 41.6±2.2 0.156 -0.65±0.56 Contains impurities 46.4±3.8 0.064 -1±0.2

[0452] Based on the monoisotopic mass of the impurity, the impure mRNA-LNP was determined to be an incompletely deprotected form of ALC-0315 containing an O-Boc group ( Figure 7B). Mildly acid-labile protecting groups were used in the synthesis of ALC-0315. The impurity peak was subjected to deprotection under acidic conditions and then reanalyzed by LC-MS. Deprotection successfully converted most of the impurity fraction to ALC-0315, thus confirming its identity ( Figure 8 ).

[0453] Summarize

[0454] Although R measured by bulk DLS h Can be used as an indicator of LNP stability, which can lead to misleading results, especially when studying LNPs in complex media such as human serum or plasma. High molecular weight plasma or serum components greatly interfere with DLS, so dilute plasma solutions or LNP recovery techniques such as ultracentrifugation are required. Unfortunately, dilute solutions cannot fully reproduce the results of undiluted physiological fluids, and LNP recovery techniques are prone to deviations because they tend to recover larger particles. Therefore, important degradation processes may even be overlooked. In addition, the use of DLS may be problematic because it can only provide relative size distribution information and cannot provide the accurate number of intact LNPs remaining over time.

[0455] The SEC-MALS method described herein provides a method for measuring LNP stability in complex media with improved accuracy. After LNP is separated from interfering plasma or serum components by SEC, MALS can measure the quantity of remaining complete LNP with high sensitivity based on their retention time. The LNP of Cominarty lipid composition degrades relatively fast (half-life of 80 to 85min) in both plasma and serum. MALS also reveals that LNP lipids are progressively replaced by plasma components, and LC-MS / MS confirms this point. The method is also proven to be a useful quality control tool because it detects the destabilizing effect of ALC-0315 impurities protected by Boc on mRNA-LNP. SEC-MALS can be used for lipid formulation optimization and quality control to accelerate the discovery of therapy.

[0456] Compared to measuring nanoparticle degradation using fluorescently labeled nanoparticles (including phospholipid bilayers stabilized with Alexa Fluor 488 via an apolipoprotein scaffold), which uses a fluorescence detector combined with SEC to measure the amount of intact LNPs over time after incubation in serum, the method described herein is advantageous, at least in part, because the fluorescent label can affect the stability of the nanoparticles, while MALS is a highly sensitive label-free technique.

[0457] Incorporated by Reference

[0458] All publications, patents, and accession numbers mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and specifically indicated to be incorporated by reference.

[0459] Equivalent

[0460] Although specific embodiments of the subject invention have been discussed, the above description is illustrative rather than restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims and their full scope of equivalents, as well as the specification and variations thereof.

Claims

1. A method for determining the stability of lipid nanoparticles (LNPs) in a subject, wherein include: obtaining a first sample comprising the LNP from the subject; performing size exclusion chromatography (SEC) on the first sample; obtaining a multi-angle light scattering (MALS) signal from the first sample; obtaining a second sample comprising the LNP from the subject; performing SEC on the second sample; obtaining a second MALS signal from the second sample; as well as comparing the first MALS signal to the second MALS signal, wherein the first and second samples comprise physiological fluids, and wherein a comparison between the first MALS signal and the second MALS signal is indicative of stability of the LNP in the subject, The stability of the LNP in the subject is thereby determined.

2. The method of claim 1, wherein the SEC is performed in a dual column configuration using a first column having a first average pore size and a second column having a second average pore size, and wherein the first average pore size is larger than the second average pore size.

3. The method of claim 2, wherein the first average pore size has a molecular weight cutoff (MWCO) of 1 MDa to 20 MDa, and wherein the second average pore size has a MWCO of 0.1 MDa to 10 MDa.

4. The method of claim 1, wherein the SEC is performed using a polymer matrix column having a length of about 100 mm to 1000 mm with a biocompatible buffer system.

5. The method of claim 1, wherein the MALS is performed at scattering angles of 15° and 90° and using a laser wavelength of about 500 nm to 800 nm.

6. The method of claim 1, wherein the MALS is performed using a sample cell volume of about 1 μL to 50 μL and a scattering volume of about 0.001 μL to 0.1 μL.

7. The method of claim 1, wherein the MALS is performed in a temperature range of about 20°C to 70°C with a temperature stability of no more than ±1°C.

8. The method of claim 1, wherein the MALS is performed at a pH range of about 1-12.

9. The method of claim 1, wherein the size of the LNP is determined.

10. The method of claim 1, wherein the molecular weight (MW) of the LNP is determined.

11. The method of claim 1, wherein the half-life of the LNP is determined.

12. The method of claim 1, wherein the method does not include the step of recovering the LNPs by ultracentrifugation.

13. The method of claim 1, wherein the method does not include the step of diluting the sample to remove high molecular weight plasma or serum components.

14. The method of claim 1, wherein the method does not include the step of labeling the LNP with a fluorophore.

15. The method of claim 1, wherein the LNP is loaded with nucleic acid.

16. The method of claim 1, wherein the nucleic acid is mRNA, small interfering RNA (siRNA), antisense oligonucleotide (ASO), or DNA.

17. The method of claim 16, wherein the physiological fluid is plasma, serum or blood.

18. A system comprising a processor configured to perform the method of claim 1.

19. A method for determining the purity of lipid nanoparticles (LNP) in a sample, wherein include: obtaining a first aliquot of the sample; subjecting the first aliquot to size exclusion chromatography (SEC); obtaining a multi-angle light scattering (MALS) signal from the first aliquot; obtaining a second aliquot of the sample; performing SEC on the second aliquot; obtaining a second MALS signal from the second aliquot; as well as comparing the first MALS signal to the second MALS signal to determine the stability of the LNP in the sample, which is indicative of the purity of the LNP in the sample, wherein the sample comprises a physiological fluid, The purity of the LNPs in the sample is thereby determined.

20. A method for monitoring a process for preparing lipid nanoparticles (LNPs), wherein include: obtaining a first sample comprising the LNPs from a process for preparing the LNPs; performing size exclusion chromatography (SEC) on the first sample; as well as obtaining a multi-angle light scattering (MALS) signal from the first sample, obtaining a second sample comprising the LNPs from the process of preparing the LNPs; performing SEC on the second sample; as well as obtaining a second MALS signal from the second sample, The first and second samples comprise physiological fluids, thereby monitoring the process of preparing the LNP.

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