Lipid nanoparticle compositions and methods of formulating the same
Provided herein are compositions and methods of reducing adduct formation.
1 . A method of administering mRNA to a subject, comprising administering to the subject a composition comprising a lipid nanoparticle comprising a mRNA, a phospholipid, a cholesterol, a PEG-lipid, and an ionizable lipid,
wherein the mRNA comprises an N1-methyl-pseudouridine (m1ψ) nucleobase,
wherein the ionizable lipid is selected from
wherein the phospholipid comprises one or more selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin,
wherein the PEG-lipid comprises one or more selected from 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA),
wherein the composition comprises a Tris (tris(hydroxymethyl)aminomethane) buffer and sucrose, and
wherein less than about 10% of the mRNA is in the form of an ionizable lipid-polynucleotide adduct impurity, as measured by reverse phase ion pair high performance liquid chromatography (RP-IP HPLC).
2 . The method of claim 1 , wherein the ionizable lipid is
3 . The method of claim 2 , wherein less than about 5% of the mRNA in the composition is in the form of the ionizable lipid-polynucleotide adduct impurity.
4 . The method of claim 2 , wherein less than about 1% of the mRNA in the composition is in the form of the ionizable lipid-polynucleotide adduct impurity.
5 . The method of claim 2 , wherein an amount of the ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 2% per day when the composition is stored at a temperature of about 25° C. or below.
6 . The method of claim 2 , wherein an amount of the ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when the composition is stored at a temperature of about 5° C. or below.
7 . The method of claim 2 , wherein an amount of the ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when the composition is stored at a refrigerated temperature.
8 . The method of claim 7 , wherein the refrigerated temperature is about 5° C.
9 . The method of claim 2 , wherein the ionizable lipid-polynucleotide adduct impurity comprises an aldehyde-mRNA adduct impurity.
10 . The method of claim 2 , wherein an amount of lipid aldehydes in the composition is less than about 50 ppm.
11 . The method of claim 2 , wherein the composition comprises a molar ratio of 20-60% ionizable lipid, 5-25% phospholipid, 25-55% cholesterol, and 0.5-15% PEG lipid, based on the lipid components.
12 . The method of claim 1 , wherein the ionizable lipid is
13 . The method of claim 12 , wherein less than about 5% of the mRNA in the composition is in the form of the ionizable lipid-polynucleotide adduct impurity.
14 . The method of claim 12 , wherein less than about 1% of the mRNA in the composition is in the form of the ionizable lipid-polynucleotide adduct impurity.
15 . The method of claim 12 , wherein an amount of the ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 2% per day when the composition is stored at a temperature of about 25° C. or below.
16 . The method of claim 12 , wherein an amount of the ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when the composition is stored at a temperature of about 5° C. or below.
17 . The method of claim 12 , wherein an amount of the ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when the composition is stored at a refrigerated temperature.
18 . The method of claim 17 , wherein the refrigerated temperature is about 5° C.
19 . The method of claim 12 , wherein the ionizable lipid-polynucleotide adduct impurity comprises an aldehyde-mRNA adduct impurity.
20 . The method of claim 12 , wherein an amount of lipid aldehydes in the composition is less than about 50 ppm.
21 . The method of claim 12 , wherein the composition comprises a molar ratio of 20-60% ionizable lipid, 5-25% phospholipid, 25-55% cholesterol, and 0.5-15% PEG lipid, based on the lipid components.
22 . The method of claim 1 , wherein the phospholipid comprises one or more selected from distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylcholine (DPPC), and wherein the PEG-lipid comprises one or more selected from 1,2-dimyristoyl-rac-glycerol-methoxy (polyethylene glycol)-2000 (DMG-PEG2000) and 1,2-distearoyl-sn-glycerol-methoxy (polyethylene glycol)-2000 (DSPE-PEG2000).