Stabilized formulations of lipid nanoparticles
AssigneeModernaTX, Inc.
InventorMark BRADER
Life of this patent
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Abstract
The disclosure features a lipid nanoparticle (LNP) formulation comprising a plurality of LNPs and a stabilizing agent that mitigates the degradation of the LNPs or a subpopulation thereof. Lipid nanoparticles further including therapeutics and/or prophylactics such as RNA are useful in the delivery of therapeutics and/or prophylactics to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression. Methods of manufacturing LNP formulations and screening for a stabilizing agent are also disclosed.
Claims166
A lipid nanoparticle (LNP) formulation comprising a plurality of LNPs and a stabilizing agent that mitigates the degradation of the LNPs or a subpopulation thereof, wherein each of the LNPs comprises an ionizable lipid and a structural lipid, and the stabilizing agent comprises a surfactant, a cryoprotectant, a chelator, an antioxidant, or any combination thereof.
The formulation of any one of the preceding claims, wherein the formulation is an aqueous formulation or a frozen formulation thereof.
The formulation of any one of the preceding claims, wherein the degradation comprises a phase separation of one or more LNP components from the remainder of LNP.
The formulation of any one of the preceding claims, wherein the degradation comprises a phase separation of a fraction of the structure lipid, a phase separation of a fraction of the ionizable lipid, or both from the remainder of LNP.
The formulation of any one of the preceding claims, wherein the formulation has a decreased fraction of the phase-separated structure lipid, a decreased fraction of the phase-separated ionizable lipid, or both, as compared to a corresponding formulation which does not comprise the stabilizing agent.
The formulation of any one of the preceding claims, wherein the fraction of the phase-separated structure lipid is between about 0.1% and 50% of the total amount of the structure lipid in the formulation or the fraction of the phase-separated ionizable lipid is between about 0.1% and 50% of the total amount of the ionizable lipid in the formulation.
The formulation of any one of the preceding claims, wherein the degradation yields an increased average size of particles in the formulation.
The formulation of any one of the preceding claims, wherein the formulation has a decreased average size of particles as compared to a corresponding formulation which does not comprise the stabilizing agent.
The formulation of any one of the preceding claims, wherein the the average size of particles in the formulation is about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, or about 30% or less, as compared to that of a corresponding formulation which does not comprise the stabilizing agent.
The formulation of any one of the preceding claims, wherein decrease in the fraction of the phase-separated structure lipid or in the fraction of the phase-separated ionizable lipid is about 20% or more as compared to that of a corresponding formulation which does not comprise the stabilizing agent.
The formulation of any one of the preceding claims, wherein the degradation comprises a formation of reduced-size particles from the LNPs or the subpopulation thereof.
The formulation of any one of the preceding claims, wherein the average size of the reduced-size particles is smaller than the average size of the LNPs or the subpopulation thereof.
The formulation of any one of the preceding claims, wherein the formation of the reduced-size particles yields an increased number of particles in the formulation.
The formulation of any one of the preceding claims, wherein the formulation has a decreased number of particles as compared to a corresponding formulation which does not comprise the stabilizing agent.
The formulation of any one of the preceding claims, wherein the number of particles in the formulation is about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, or about 30% or less, as compared to that of a corresponding formulation which does not comprise the stabilizing agent.
The formulation of any one of the preceding claims, wherein the cryoprotectant comprises ethanol.
The formulation of any one of the preceding claims, wherein the cryoprotectant comprises a polyol.
The formulation of any one of the preceding claims, wherein the cryoprotectant comprises a diol or a triol.
The formulation of any one of the preceding claims, wherein the cryoprotectant comprises propylene glycol.
The formylation of any one of the preceding claims, wherein the cryoprotectant comprises buranediol, diethylene glycol, or polyethylene glycol (PEG).
The formylation of any one of the preceding claims, wherein the cryoprotectant comprises dimethyl sulfoxide (DMSO) or 3-(1-Pyridinio)-1-propanesulfonate (NDSB-201).
The formylation of any one of the preceding claims, wherein the cryoprotectant comprises lithium acetate, lithium chloride, lithium formate, lithium nitrate, magnesium acetate, sodium chloride, or any hydrate thereof.
The formulation of any one of the preceding claims, wherein the cryoprotectant comprises glycerol.
The formulation of any one of the preceding claims, wherein the cyoprotectant comprises glycerol and ethanol.
The formulation of any one of the preceding claims, wherein the concentration of glycerol in the formulation ranges from about 0.05% w/v to about 50% w/v, from about 0.05% w/v to about 25% w/v, from about 0.5% w/v to 10% w/v, or from about 1% w/v to about 5% w/v.
The formulation of any one of the preceding claims, wherein the concentration of glycerol in the formulation ranges from about 1.5% w/v to about 3% w/v.
The formulation of any one of the preceding claims, wherein the concentration of glycerol in the formulation ranges from about 2% w/v to about 2.5% w/v.
The formulation of any one of the preceding claims, wherein the concentration of ethanol in the formulation ranges from about 0.05% w/v to about 50% w/v, from about 0.05% w/v to about 25% w/v, from about 0.5% w/v to 10% w/v, or from about 1% w/v to about 5% w/v.
The formulation of any one of the preceding claims, wherein the concentration of ethanol in the formulation ranges from about 0.7% w/v to about 1.5% w/v.
The formulation of any one of the preceding claims, wherein the concentration of ethanol in the formulation ranges from about 1% w/v to about 1.2% w/v.
The formulation of any one of the preceding claims, wherein the weight ratio of glycerol and ethanol in the formulation ranges from about 1:25 to about 100:1 by weight, from about 1:10 to about 40:1 by weight, from about 1:5 to about 20:1 by weight, or from about 1:1 to about 4:1 by weight.
The formulation of any one of the preceding claims, wherein the weight ratio of glycerol and ethanol in the formulation is about 2:1 by weight.
The formulation of any one of the preceding claims, wherein:
- the concentration of glycerol in the formulation ranges from about 2% w/v to about 2.5% w/v;
- and
- the concentration of ethanol in the formulation ranges from about 1% w/v to about 1.2% w/v.
The formulation of any one of the preceding claims, wherein:
- the concentration of glycerol in the formulation is about 2.3% w/v;
- and
- the concentration of ethanol in the formulation is about 1.2% w/v.
The formulation of any one of the preceding claims, wherein the concentration of glycerol in the formulation ranges from about 1 mM to about 10 M, from about 5 mM to about 5 M, from about 10 mM to about 1 M, from about 50 mM to about 600 mM, from about 100 mM to about 500 mM, or from about 200 mM to about 300 mM.
The formulation of any one of the preceding claims, wherein the concentration of glycerol in the formulation is about 250 mM.
The formulation of any one of the preceding claims, wherein the concentration of ethanol in the formulation ranges from about 1 mM to about 10 M, from about 5 mM to about 5 M, from about 10 mM to about 1 M, from about 50 mM to about 600 mM, from about 100 mM to about 500 mM, or from about 200 mM to about 300 mM
The formulation of any one of the preceding claims, wherein the concentration of ethanol in the formulation is about 250 mM.
The formulation of any one of the preceding claims, wherein the molar ratio of glycerol and ethanol in the formulation ranges from about 1:100 to about 100:1 by weight, from about 1:50 to about 50:1, from about 25:1 to about 1:25, from about 10:1 to about 1:10, from about 5:1 to about 1:5, or from about 2:1 to about 1:2.
The formulation of any one of the preceding claims, wherein the molar ratio of glycerol and ethanol in the formulation is about 1:1.
The formulation of any one of the preceding claims, wherein:
- the concentration of glycerol in the formulation ranges from about 200 mM to about 300 mM;
- and
- the concentration of ethanol in the formulation ranges from ranges from about 200 mM to 5 about 300 mM.
The formulation of any one of the preceding claims, wherein:
- the concentration of glycerol in the formulation is about 250 mM;
- and
- the concentration of ethanol in the formulation is about 250 mM.
The formulation of any one of the preceding claims, wherein the surfactant comprises a nonionic surfactant.
The formulation of any one of the preceding claims, wherein the surfactant comprises a polysorbate.
The formulation of any one of the preceding claims, wherein the polysorbate has a structure of Formula (IV):
- wherein the sum of w, x, y, and z ranges from about 0 to about 200.
The formulation of any one of the preceding claims, wherein the sum of w, x, y, and z ranges from about 0 to about 100, from about 10 to about 90, or from about 20 to about 80.
The formulation of any one of the preceding claims, wherein the sum of w, x, y, and z is about 20, about 40, about 60, or about 80.
The formulation of any one of the preceding claims, wherein the polysorbate is polysorbate 20, polysorbate 40, polysorbate, 60, or polysorbate 80.
The formulation of any one of the preceding claims, wherein the polysorbate is polysorbate 20 or polysorbate 80.
The formulation of any one of the preceding claims, wherein the polysorbate is polysorbate 20.
The formulation of any one of the preceding claims, wherein the concentration of polysorbate 20 ranges from about 0.0001% w/v to about 1.0% w/v, from about 0.001% w/v to about 0.1% w/v, from about 0.005% w/v to about 0.05% w/v, from about 0.008% w/v to about 0.03% w/v, or from about 0.009% w/v to about 0.02% w/v.
The formulation of any one of the preceding claims, wherein the concentration of polysorbate 20 is about 0.01% w/v.
The formulation of any one of the preceding claims, wherein the stabilizing agent comprises polysorbate, glycerol, and ethanol.
The formulation of any one of the preceding claims, wherein the stabilizing agent comprises polysorbate 20, glycerol, and ethanol.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation ranges from about 0.009% w/v to about 0.02% w/v;
- the concentration of glycerol in the formulation ranges from about 2% w/v to about 2.5% w/v;
- and
- the concentration of ethanol in the formulation ranges from about 1% w/v to about 1.5% w/v.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation ranges from about 0.009% w/v to about 0.02% w/v;
- the concentration of glycerol in the formulation is about 2.3% w/v;
- and
- the concentration of ethanol in the formulation is about 1.2% w/v.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation is about 0.01% w/v;
- the concentration of glycerol in the formulation ranges from about 2% w/v to about 2.5% w/v;
- and
- the concentration of ethanol in the formulation ranges from about 1% w/v to about 1.5% w/v.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation is about 0.01% w/v;
- the concentration of glycerol in the formulation is about 2.3% w/v;
- and
- the concentration of ethanol in the formulation is about 1.2% w/v.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation ranges from about 0.009% w/v to about 0.02% w/v;
- the concentration of glycerol in the formulation ranges from about 200 mM to about 300 mM;
- and
- the concentration of ethanol in the formulation ranges from about 200 mM to about 300 mM.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation ranges from about 0.009% w/v to about 0.02% w/v;
- the concentration of glycerol in the formulation is about 250 mM;
- and
- the concentration of ethanol in the formulation is about 250 mM.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation is about 0.01% w/v;
- the concentration of glycerol in the formulation ranges from about 200 mM to about 300 mM;
- and
- the concentration of ethanol in the formulation ranges from about 200 mM to about 300 mM.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation is about 0.01% w/v;
- the concentration of glycerol in the formulation is about 250 mM;
- and
- the concentration of ethanol in the formulation is about 250 mM.
The formulation of any one of the preceding claims, wherein the pH value of the formulation ranges from about 4 to about 11, from about 5 to about 10, from about 6 to about 9, from about 7 to about 8, or from about 7.3 to about 7.5.
The formulation of any one of the preceding claims, wherein the pH value of the formulation is about 7.4.
The formulation of any one of the preceding claims, wherein:
- the concentration of polysorbate in the formulation is about 0.01% w/v;
- the concentration of glycerol in the formulation is about 250 mM;
- the concentration of ethanol in the formulation is about 250 mM;
- and
- the pH value of the formulation is about 7.4.
The formulation of any one of the preceding claims, wherein the formulation is free of one or more salts selected from the group consisting of lithium salts, magnesium salts, and magnesium salts.
The formulation of any one of the preceding claims, wherein the formulation is free of one or more sodium salts.
The formulation of any one of the preceding claims, wherein the formulation is free of sodium chloride.
The formulation of any one of the preceding claims, wherein the chelator comprises diethylenetriamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), iminodisuccinic acid, polyaspartic acid, ethylenediamine-N,N′-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), L-glutamic acid N,N-diacetic acid (GLDA), a salt thereof, or any combination thereof.
The formulation of any one of the preceding claims, wherein the antioxidant comprises ascorbic acid, citric acid, malic acid, methionine, monothioglycerol, phosphoric acid, potassium metabisulfite, sodium metabisulfite, alpha-tocopherol, or any combination thereof.
The formulation of any one of the preceding claims, wherein the concentration of the cryoprotectant in the formulation ranges from about 0.05% to about 50% by weight.
The formulation of any one of the preceding claims, wherein the concentration of the cryoprotectant in the formulation ranges from about 0.05% to about 25% by weight, from about 1% to 15% by weight, or from about 3% to about 12.5% by weight.
The formulation of any one of the preceding claims, wherein the concentration of the cryoprotectant in the formulation ranges from about 1% to about 8% by weight.
The formulation of any one of the preceding claims, wherein the concentration of the cryoprotectant in the formulation ranges from about 2% to about 7% by weight.
The formulation of any one of the preceding claims, wherein the degradation of LNPs is determined by dynamic light scattering (DLS).
The formulation of any one of the preceding claims, wherein the degradation of LNPs is determined by nanoparticle tracking analysis (NTA).
The formulation of any one of the preceding claims, wherein the degradation of LNPs is determined by turbidity analysis.
The formulation of any one of the preceding claims, wherein the degradation of LNPs is determined by flow microscopy analysis.
The formulation of any one of the preceding claims, wherein the degradation of LNPs is determined by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), turbidity analysis, flow microscopy analysis, flow cytometry, FTIR microscopy, liquid chromatography (LC), resonant mass measurement (RMM), Raman microscopy, filtration, laser diffraction, electron microscopy, atomic force microscopy (AFM), static light scattering (SLS), multi-angle static light scattering (MALS), field flow fractionation (FFF), analytical ultracentrifugation (AUC), or any combination thereof.
The formulation of any one of the preceding claims, wherein the formulation has an increase in LNP mean particle size of about 20% or less after storage at about −20° C. or lower for at least one month.
The formulation of any one of the preceding claims, wherein the formulation has an increase in LNP mean particle size of about 20% or less after up to 30 freeze/thaw cycles.
The formulation of any one of the preceding claims, wherein the formulation has an increase in turbidity of about 20% or less after storage at about −20° C. or lower for at least one month.
The formulation of any one of the preceding claims, wherein the formulation has an increase in turbidity of about 20% or less after up to 30 freeze/thaw cycles.
The formulation of any one of the preceding claims, wherein the formulation is purified prior to storage at about −20° C. or lower.
The formulation of any one of the preceding claims, wherein the formulation is purified by filtration.
The formulation of any one of the preceding claims, wherein the formulation is substantially free of impurities.
The formulation of any one of the preceding claims, wherein the formulation contains about 20% or less, about 10% or less, about 5% or less, about 1% or less, or about 0.5% or less of impurities.
The formulation of any one of the preceding claims, wherein the impurities comprise sub-visible particulates.
The formulation of any one of the preceding claims further comprising a sugar.
The formulation of any one of the preceding claims, wherein the sugar is disaccharide.
The formulation of any one of the preceding claims, wherein the sugar is sucrose or trehalose or a combination thereof.
The formulation of any one of the preceding claims, wherein the total concentration of the sugar ranges between 0% and about 30% by weight.
The formulation of any one of the preceding claims further comprising a salt.
The formulation of any one of the preceding claims, wherein the salt is a chloride salt, preferably NaCl.
The formulation of any one of the preceding claims, wherein the formulation further comprises a buffer.
The formulation of any one of the preceding claims, wherein the buffer comprises Tris and the formulation is optionally a frozen formulation.
The formulation of any one of the preceding claims, wherein the buffer comprises a phosphate and the formulation is optionally a non-frozen formulation.
The formulation of any one of the preceding claims, wherein the formulation has a pH value ranging between about 4 and about 8 prior to freezing.
The formulation of any one of the preceding claims, further comprising one or more therapeutic and/or prophylactic agents carried by the LNPs.
The formulation of any one of the preceding claims, wherein the therapeutic and/or prophylactic agents comprise a nucleic acid.
The formulation of any one of the preceding claims, wherein the formulation has about 0.025 mg/mL to about 4 mg/mL of the nucleic acid, preferably about 0.025 mg/mL to about 0.4, 0.05 mg/mL to about 0.2 mg/mL, or 0.05 mg/mL to about 0.1 mg/mL of the nucleic acid prior to freezing.
The formulation of any one of the preceding claims, wherein the therapeutic and/or prophylactic agent is a ribonucleic acid (RNA).
The formulation of any one of the preceding claims, wherein the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), a long non-coding RNA (lncRNA) and mixtures thereof.
The formulation of any one of the preceding claims, wherein the RNA is an mRNA.
The formulation of any one of the preceding claims, wherein the mRNA includes one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and/or a 5′ cap structure.
The formulation of any one of the preceding claims, wherein the mRNA is at least 30 nucleotides in length.
The formulation of any one of the preceding claims, wherein the mRNA is at least 300 nucleotides in length.
The formulation of any one of the preceding claims, wherein the encapsulation efficiency of the therapeutic and/or prophylactic agent is at least 50%, at least 80%, at least 90%, or at least 95%.
The formulation of any one of the preceding claims, wherein the number ratio between the LNPs that carry one or more therapeutic and/or prophylactic agents and those free of any therapeutic and/or prophylactic agents is about 8:2 to about 10:0.
The formulation of any one of the preceding claims, wherein the encapsulation efficiency or the number ratio between the LNPs that carry one or more therapeutic and/or prophylactic agents and those free of any therapeutic and/or prophylactic agents is substantially the same after storage at about −20° C. or lower for at least one month.
The formulation of any one of the preceding claims, wherein the encapsulation efficiency or the number ratio between the LNPs that carry one or more therapeutic and/or prophylactic agents and those free of any therapeutic and/or prophylactic agents is substantially the same after up to 30 freeze/thaw cycles.
The formulation of any one of the preceding claims, wherein the wt/wt ratio of the LNPs to the therapeutic and/or prophylactic agents is from about 10:1 to about 60:1.
The formulation of any one of the preceding claims, wherein the N:P ratio is from about 2:1 to about 30:1.
The formulation of any one of the preceding claims, wherein the mean size of the LNPs is from about 70 nm to about 100 nm.
The formulation of any one of the preceding claims, wherein the formulation has little or no immunogenicity.
The formulation of any one of the preceding claims, wherein the formulation further comprises a therapeutic or prophylactic agent and has an increased therapeutic index as compared to a corresponding formulation which does not comprise the stabilizing agent.
The formulation of any one of the preceding claims, wherein the LNPs further comprise a neutral lipid.
The formulation of any one of the preceding claims, wherein the neutral lipid is a phospholipid or an analog or derivative thereof.
The formulation of any one of the preceding claims, wherein the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 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-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 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), sphingomyelin, and mixtures thereof.
The formulation of any one of the preceding claims, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.
The formulation of any one of the preceding claims, wherein the LNPs further comprises a PEG lipid.
The formulation of any one of the preceding claims, wherein the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
The formulation of any one of the preceding claims, wherein the LNPs comprise about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol % to about 10 mol % PEG lipid.
The formulation of any one of the preceding claims, wherein the LNPs comprise about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % PEG lipid.
The formulation of any one of the preceding claims, wherein the ionizable lipid comprises an ionizable amino lipid.
The formulation of any one of the preceding claims, wherein the ionizable lipid comprises a compound of any of Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId) and (IIe).
The formulation of any one of the preceding claims, wherein the LNPs further comprise a cationic and/or ionizable lipid selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(β3)-cholest-5-en-3-yloxy] octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(313)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[(β3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)), (12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, and N,N-dimethyl-1-{(1S,2R)-2-octylcyclopropyl}heptadecan-8-amine.
A method of mitigating degradation of lipid nanoparticles in a lipid nanoparticle (LNP) formulation, the method comprising adding a stabilizing agent to a first LNP formulation to form a second LNP formulation before or during storage −20° C. or lower or before a freeze/thaw cycle, wherein each of the LNPs comprises an ionizable lipid and a structural lipid, and the stabilizing agent comprises a cryoprotectant, a chelator, an antioxidant, or any combination thereof.
A method of producing a stabilized lipid nanoparticle (LNP) formulation, comprising mixing a stabilizing agent with a first LNP formulation to form a second LNP formulation, wherein each of the LNPs comprises an ionizable lipid and a structural lipid, and the stabilizing agent comprises a cryoprotectant, a chelator, an antioxidant, or any combination thereof such that the stabilizing agent mitigates degradation of the LNPs or a subpopulation thereof.
A method of screening for a stabilizing agent for mitigating degradation of lipid nanoparticles in a lipid nanoparticle (LNP) formulation, the method comprising:
- (a) providing a first LNP formulation absent a stabilizing agent and a second LNP formulation comprising the stabilizing agent, wherein the first and second LNP formulations are identical except for the stabilizing agent and each of the LNPs in the first and second LNP formulations comprises an ionizable lipid and a structural lipid;
- (b) determining the degradation of the LNPs in the first and second LNP formulations upon storage at about −20° C. or lower for a period of time or upon one or more freeze/thaw cycles;
- and
- (c) selecting the stabilizing agent if the degradation of the second LNP formulation is less than that of the first LNP formulation.
The method of any one of the preceding claims, wherein the stabilizing agent comprises a cryoprotectant, a chelator, an antioxidant, or any combination thereof.
The method of any one of the preceding claims, wherein the first or the second LNP formulation further comprises a nucleic acid.
The method of any one of the preceding claims, wherein the number ratio between the LNPs that carry one or more nucleic acids and those free of any nucleic acids is about 8:2 to about 10:0.
The method of any one of the preceding claims, wherein the second LNP formulation is substantially free of impurities.
The method of any one of the preceding claims, wherein the second LNP formulation contains fewer impurities as compared to the first LNP formulation.
The method of any one of the preceding claims, wherein the second LNP formulation contains about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 1% or less, or about 0.5% or less of impurities.
The method of any one of the preceding claims, wherein the second LNP formulation has a decrease in the amount of sub-visible particulates as compared to the first LNP formulation.
The method of any one of the preceding claims, wherein the concentration of the cryoprotectant in the second LNP formulation ranges from about 0.5% to about 50% by weight, preferably from about 0.05% to about 25% by weight, from about 1% to 15% by weight, or from about 3% to about 12.5% by weight.
The method of any one of the preceding claims, wherein the formulation further comprises a salt.
The method of any one of the preceding claims, wherein the salt is a chloride salt, preferably NaCl.
The method of any one of the preceding claims, wherein the formulation further comprises a buffer.
The method of any one of the preceding claims, wherein the buffer comprises Tris and the formulation is optionally a frozen formulation.
The method of any one of the preceding claims, wherein the buffer comprises a phosphate and the formulation is optionally a non-frozen formulation.
The method of any one of the preceding claims, further comprising purifying the first LNP formulation prior to adding the stabilizing agent.
The method of any one of the preceding claims, wherein the purification comprises filtration.
The method of any one of the preceding claims, wherein the degradation comprises a phase separation of one or more LNP components from the remainder of LNP.
The method of any one of the preceding claims, wherein the degradation comprises a phase separation of a fraction of the structure lipid, a phase separation of a fraction of the ionizable lipid, or both, from the remainder of LNP.
The method of any one of the preceding claims, wherein the formulation has a decreased fraction of the phase-separated structure lipid, a decreased fraction of the phase-separated ionizable lipid, or both, as compared to a corresponding formulation which does not comprise the stabilizing agent.
The method of any one of the preceding claims, wherein the fraction of the phase-separated structure lipid is between about 0.1% and 50% of the total amount of the structure lipid in the formulation or the fraction of the phase-separated ionizable lipid is between about 0.1% and 50% of the total amount of the ionizable lipid in the formulation.
The method of any one of the preceding claims, wherein decrease in the fraction of the phase-separated structure lipid or in the fraction of the phase-separated ionizable lipid is about 20% or more as compared to that of a corresponding formulation which does not comprise the stabilizing agent.
The method of any one of the preceding claims, wherein the second LNP formulation has substantially no increase in LNP mean size after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has an increase in LNP mean size of about 20% or less after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has an increase in LNP mean size of about 20% or less after up to 30 freeze/thaw cycles.
The method of any one of the preceding claims, wherein the second LNP formulation has substantially no increase in turbidity after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has an increase in turbidity of about 20% or less after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has an increase in turbidity of about 20% or less after up to 30 freeze/thaw cycles.
The method of any one of the preceding claims, wherein the second LNP formulation has substantially no increase in polydispersity index after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has an increase in polydispersity index of about 20% or less after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has an increase in polydispersity index of about 20% or less after up to 30 freeze/thaw cycles.
The method of any one of the preceding claims, wherein the second LNP formulation has substantially no decrease in encapsulation efficiency after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has substantially no decrease in encapsulation efficiency after up to 30 freeze/thaw cycles.
The method of any one of the preceding claims, wherein the second LNP formulation has a decrease in encapsulation efficiency of about 20% or less after storage at about −20° C. or lower for at least one month.
The method of any one of the preceding claims, wherein the second LNP formulation has a decrease in encapsulation efficiency of about 20% or less after up to 30 freeze/thaw cycles.
A method of lowering immunogenicity comprising introducing the formulation of any one of the preceding claims into cells, wherein the formulation reduces the induction of the cellular immune response of the cells to the formulation, as compared to the induction of the cellular immune response in cells induced by a corresponding formulation which does not comprise the stabilizing agent.
The method of any one of the preceding claims, wherein the cellular immune response is an innate immune response, an adaptive immune response, or both.
Dependencies are inferred from the claim text.