Stabilization of lipid nanoparticle formulations

Abstract

Aspects of the present disclosure relate to compositions and methods for improving the stability of lipid nanoparticles (LNPs). In some embodiments, the LNPs contain one or more active pharmaceutical ingredients (APIs) encapsulated therein. The present disclosure is based, in part, on compositions that directly or indirectly reduce degradation (e.g., oxidation, hydrolysis, etc.) of one or more lipid components of the lipid nanoparticles. In some embodiments, the compositions contain a histidine buffer. The present disclosure also provides methods for storing the compositions contemplated herein, as well as methods for improving the stability of APIs.

Description

[Technical Field]

[0001] (Related Applications) This application claims the benefit under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Patent Application No. 63 / 435,024, entitled "STABILIZATION OF LIPID NANOPARTICLE FORMULATIONS," filed December 23, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Lipid nanoparticle (LNP) formulations are useful drug delivery systems for encapsulating various active pharmaceutical ingredients (APIs), such as RNA. LNP formulations generally contain an aqueous buffer, such as a phosphate buffer. However, current LNP formulations are susceptible to degradation and aggregate formation during storage at room temperature, and therefore, improvements are needed. Summary of the Invention

[0003] Aspects of the present disclosure relate to compositions and methods for improving the stability of lipid nanoparticles (LNPs) and one or more active pharmaceutical ingredients (APIs) encapsulated therein. The present disclosure is based, in part, on compositions that directly or indirectly reduce degradation (e.g., oxidation, hydrolysis, etc.) of one or more lipid components of lipid nanoparticles. In some embodiments, the compositions include a histidine buffer. In some embodiments, the histidine buffer inhibits oxidation and / or hydrolysis of one or more lipids of the LNPs and / or improves the stability of APIs encapsulated within the LNPs. The present disclosure also provides methods for storing the compositions contemplated herein.

[0004] Accordingly, in some aspects, the present disclosure provides pharmaceutical compositions comprising lipid nanoparticles (LNPs) comprising one or more ionizable lipids and a histidine buffer having a histidine concentration ranging from 5 mM to 30 mM and a pH ranging from about 5.0 to about 7.5.

[0005] In some embodiments, the histidine buffer concentration ranges from 10 mM to 20 mM histidine. In some embodiments, the histidine buffer concentration is at least 10 mM. In some embodiments, the histidine buffer concentration is 20 mM.

[0006] In some embodiments, the pH of the histidine buffer is a pH of about 6.0.

[0007] In some embodiments, the histidine buffer further comprises one or more salts. In some embodiments, the one or more salts comprise NaCl. In some embodiments, the histidine buffer comprises one or more non-ionic excipients, such as sucrose.

[0008] In some embodiments, one or more ionizable lipids comprise an unsaturated tail, and further optionally, one or more ionizable lipids comprise DLin-MC3-DMA (MC3) and / or dioleoyl-3-trimethylammonium propane (DOTAP). In some embodiments, one or more ionizable lipids consist of MC3 or DOTAP.

[0009] In some embodiments, the LNP comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise RNA. In some embodiments, the RNA is mRNA, siRNA, dsRNA, or miRNA. In some embodiments, the RNA is siRNA.

[0010] In some embodiments, the composition is not refrigerated or frozen (e.g., after the addition of the histidine buffer). In some embodiments, the composition is stored at a temperature above 4°C. In some embodiments, the temperature ranges from about 5°C to about 30°C. In some embodiments, the composition is frozen (e.g., before or after the addition of the histidine buffer), then thawed and stored at a temperature ranging from about 5°C to about 30°C.

[0011] In some aspects, the present disclosure provides a container containing the pharmaceutical composition described herein.In some embodiments, the container is a cartridge, a pre-filled syringe, or a glass vial.In some embodiments, the container is a pre-filled syringe or a glass vial.In some embodiments, the container is a polymer vial.

[0012] In some aspects, the present disclosure provides methods for improving the chemical stability of a lipid nanoparticle (LNP) pharmaceutical composition, the method comprising obtaining a non-histidine-buffered LNP pharmaceutical composition comprising a non-histidine buffer, and performing a buffer exchange procedure to replace the non-histidine buffer with a histidine buffer having a pH of 5.8 to 7.5 to obtain a histidine-buffered LNP pharmaceutical composition.

[0013] In some embodiments, the histidine-buffered LNP pharmaceutical composition comprises one or more ionizable lipids (e.g., one or more ionizable lipids having unsaturated fatty acid tails). In some embodiments, the ionizable lipid comprises MC3 and / or DOTAP. In some embodiments, the ionizable lipid consists of MC3 or DOTAP.

[0014] In some embodiments, the non-histidine buffer comprises a citrate buffer. In some embodiments, the pH of the citrate buffer ranges from about 3.5 to about 5.5. In some embodiments, the pH of the citrate buffer is 5.0.

[0015] In some embodiments, the histidine-buffered LNP pharmaceutical composition comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise RNA. In some embodiments, the RNA is mRNA, siRNA, dsRNA, or miRNA. In some embodiments, the RNA is siRNA.

[0016] In some embodiments, the histidine buffer has a pH of 6.0. In some embodiments, the histidine buffer has a histidine concentration ranging from about 5 mM to about 30 mM. In some embodiments, the histidine buffer has a histidine concentration ranging from about 10 mM to 20 mM. In some embodiments, the histidine concentration is at least 10 mM. In some embodiments, the histidine concentration is 20 mM.

[0017] In some embodiments, the buffer exchange procedure comprises contacting the non-histidine-buffered LNP pharmaceutical composition with a desalting column. In some embodiments, the buffer exchange procedure comprises contacting the non-histidine-buffered LNP pharmaceutical composition with dialysis tubing or performing tangential flow filtration. In some embodiments, the buffer exchange reaction comprises contacting the non-histidine-buffered LNP pharmaceutical composition with tangential flow filtration.

[0018] In some embodiments, the buffer exchange procedure involves collecting the histidine-buffered LNP pharmaceutical composition in a container (e.g., a vial or a syringe). In some embodiments, the container is a cartridge, a pre-filled syringe, or a glass vial. In some embodiments, the vial is a glass vial. In some embodiments, the vial is a polymeric vial.

[0019] In some embodiments, the method further comprises storing the histidine-buffered LNP pharmaceutical composition at a temperature above 4° C. In some embodiments, the temperature ranges from about 5° C. to about 30° C.

[0020] In some embodiments, histidine-buffered LNP pharmaceutical compositions contain less hydrolyzed lipids compared to non-histidine-buffered LNP pharmaceutical compositions stored in phosphate buffer.

[0021] In some embodiments, histidine-buffered LNP pharmaceutical compositions contain less oxidized lipids compared to non-histidine-buffered LNP pharmaceutical compositions stored in phosphate buffer.

[0022] In some embodiments, the histidine-buffered LNP pharmaceutical composition comprises LNPs that have increased colloidal stability compared to non-histidine-buffered LNPs stored in a pharmaceutical composition comprising a phosphate buffer.

[0023] In some aspects, the present disclosure provides methods for preparing a lipid nanoparticle (LNP) pharmaceutical composition, comprising preparing a first lipid composition comprising one or more lipids, preparing a second lipid composition comprising siRNA and an ionic lipid, and mixing the first lipid composition and the second lipid composition, wherein the first lipid composition and the second lipid composition are prepared using a histidine buffer having a pH of 5.8 to 6.5 and a histidine concentration of 5 mM to 30 mM.

[0024] In some aspects, the present disclosure provides a method for preparing a lipid nanoparticle (LNP) pharmaceutical composition, the method comprising obtaining a lipid composition comprising siRNA and an ionic lipid, and mixing the lipid composition with a histidine buffer having a pH of 5.8 to 6.5 and a histidine concentration of 5 mM to 30 mM. [Brief explanation of the drawings]

[0025] [Figure 1] Representative structural features of two ionic lipids, MC3 and DOTAP, used to prepare lipid nanoparticles (LNPs) are shown. [Figure 2A] The percentage of DOTAP (Figure 2A) and MC3 (Figure 2B) lipids that remained intact over 4 weeks after storage in either glass (BT5933) or cyclic olefin polymer (COP) vials at different temperatures is shown. Briefly, DOTAP or MC3 was dissolved in ethanol at a concentration of 4 mg / mL. The solution was then diluted in 1x PBS buffer and filled into either glass or COP vials. [Figure 2B]The percentage of DOTAP (Figure 2A) and MC3 (Figure 2B) lipids that remained intact over 4 weeks after storage in either glass (BT5933) or cyclic olefin polymer (COP) vials at different temperatures is shown. Briefly, DOTAP or MC3 was dissolved in ethanol at a concentration of 4 mg / mL. The solution was then diluted in 1x PBS buffer and filled into either glass or COP vials. [Figure 3A] Representative data are shown showing the hydrolysis rate (Figure 3A) and oxidation rate (Figure 3B) of DOTAP lipids, and the hydrolysis rate of MC3 lipids (Figure 3C) over a 4-week period after storage at different temperatures in either glass (BT5933) or cyclic olefin polymer (COP) vials. [Figure 3B] Representative data are shown showing the hydrolysis rate (Figure 3A) and oxidation rate (Figure 3B) of DOTAP lipids, and the hydrolysis rate of MC3 lipids (Figure 3C) over a 4-week period after storage at different temperatures in either glass (BT5933) or cyclic olefin polymer (COP) vials. [Figure 3C] Representative data are shown showing the hydrolysis rate (Figure 3A) and oxidation rate (Figure 3B) of DOTAP lipids, and the hydrolysis rate of MC3 lipids (Figure 3C) over a 4-week period after storage at different temperatures in either glass (BT5933) or cyclic olefin polymer (COP) vials. [Figure 4A] Representative data are shown showing that storage of DOTAP lipids in histidine buffer improves the percentage of intact DOTAP lipids after storage over a range of temperatures over a 4-week period (Figure 4A), and that histidine buffer reduces hydrolysis of DOTAP lipids during storage over a range of temperatures over a 4-week period (Figure 4B). Histidine buffer has a more significant effect on the chemical stability of DOTAP than the container / closure system. [Figure 4B]Representative data are shown showing that storage of DOTAP lipids in histidine buffer improves the percentage of intact DOTAP lipids after storage over a range of temperatures over a 4-week period (Figure 4A), and that histidine buffer reduces hydrolysis of DOTAP lipids during storage over a range of temperatures over a 4-week period (Figure 4B). Histidine buffer has a more significant effect on the chemical stability of DOTAP than the container / closure system. [Figure 5A] Representative data are shown showing that storage of MC3 lipids in histidine buffer improves the percentage of intact MC3 lipids after storage over a range of temperatures over a 4-week period (Figure 5A), and that histidine buffer reduces hydrolysis (Figure 5B) and oxidation (Figure 5C) of MC3 lipids during storage over a range of temperatures over a 4-week period. Histidine buffer has a more significant effect on the chemical stability of MC3 than the container / closure system. [Figure 5B] Representative data are shown showing that storage of MC3 lipids in histidine buffer improves the percentage of intact MC3 lipids after storage over a range of temperatures over a 4-week period (Figure 5A), and that histidine buffer reduces hydrolysis (Figure 5B) and oxidation (Figure 5C) of MC3 lipids during storage over a range of temperatures over a 4-week period. Histidine buffer has a more significant effect on the chemical stability of MC3 than the container / closure system. [Figure 5C] Representative data are shown showing that storage of MC3 lipids in histidine buffer improves the percentage of intact MC3 lipids after storage over a range of temperatures over a 4-week period (Figure 5A), and that histidine buffer reduces hydrolysis (Figure 5B) and oxidation (Figure 5C) of MC3 lipids during storage over a range of temperatures over a 4-week period. Histidine buffer has a more significant effect on the chemical stability of MC3 than the container / closure system. [Figure 6A]Representative data are shown showing that storage of empty MC3-LNP in histidine buffer stabilizes LNP particle size when stored at 5°C (Figure 6A) and at room temperature (RT) for 4 weeks (Figure 6B). Histidine buffer is used in the 5933 His sample, while 1x PBS buffer is used in the other samples. BT5933 is a Type 1 glass vial with a coefficient of expansion (COE) of 51. BT5974 vial is a Type 1 glass vial with a COE of 33. [Figure 6B] Representative data are shown showing that storage of empty MC3-LNP in histidine buffer stabilizes LNP particle size when stored at 5°C (Figure 6A) and at room temperature (RT) for 4 weeks (Figure 6B). Histidine buffer is used in the 5933 His sample, while 1x PBS buffer is used in the other samples. BT5933 is a Type 1 glass vial with a coefficient of expansion (COE) of 51. BT5974 vial is a Type 1 glass vial with a COE of 33. [Figure 7A] Representative data are shown showing that storage of empty DOTAP-LNP in histidine does not substantially change LNP size compared to PBS buffer after up to 4 weeks of storage at either 5° C. or room temperature. Histidine buffer is used in the 5933 His sample, while 1× PBS buffer is used in the other samples. [Figure 7B] Representative data are shown showing that storage of empty DOTAP-LNP in histidine does not substantially change LNP size compared to PBS buffer after up to 4 weeks of storage at either 5° C. or room temperature. Histidine buffer is used in the 5933 His sample, while 1× PBS buffer is used in the other samples. [Figure 8A] Representative data are shown showing that storage of empty MC3-LNPs in histidine buffer stabilizes the percentage of intact MC3 over a range of temperatures over a 4-week period (Figure 8A) and reduces oxidation of MC3 in the LNPs (Figure 8B). [Figure 8B]Representative data are shown showing that storage of empty MC3-LNPs in histidine buffer stabilizes the percentage of intact MC3 over a range of temperatures over a 4-week period (Figure 8A) and reduces oxidation of MC3 in the LNPs (Figure 8B). [Figure 9A] Representative data are shown showing that storage of empty DOTAP-LNPs in histidine buffer stabilizes the percentage of intact DOTAP over a 4-week temperature range (Figure 9A) and reduces hydrolysis of DOTAP in DOTAP-LNPs over a 4-week temperature range (Figure 9B). [Figure 9B] Representative data are shown showing that storage of empty DOTAP-LNPs in histidine buffer stabilizes the percentage of intact DOTAP over a 4-week temperature range (Figure 9A) and reduces hydrolysis of DOTAP in DOTAP-LNPs over a 4-week temperature range (Figure 9B). [Figure 10A] Representative data are shown showing the stabilization of siRNA-loaded MC3-LNP particle size (FIG. 10A) and polydispersity index (FIG. 10B) after formulation in histidine buffer compared to formulation in PBS buffer over a range of temperatures over a 4-week period. All samples were stored in glass vials. [Figure 10B] Representative data are shown showing the stabilization of siRNA-loaded MC3-LNP particle size (FIG. 10A) and polydispersity index (FIG. 10B) after formulation in histidine buffer compared to formulation in PBS buffer over a range of temperatures over a 4-week period. All samples were stored in glass vials. [Figure 11A] Representative data are shown showing that histidine buffer storage stabilizes MC3-LNP encapsulation of siRNA as a function of time compared to PBS buffer over a 4-week temperature range (Figure 11A) and reduces subvisible particle concentration as measured by microflow imaging (MFI) after 4 weeks over a temperature range (Figure 11B). [Figure 11B]Representative data are shown showing that histidine buffer storage stabilizes MC3-LNP encapsulation of siRNA as a function of time compared to PBS buffer over a 4-week temperature range (Figure 11A) and reduces subvisible particle concentration as measured by microflow imaging (MFI) after 4 weeks over a temperature range (Figure 11B). [Figure 12A] Representative data are shown showing the relative percentage of intact MC3 in MC3-LNPs encapsulating siRNA formulated in PBS (FIG. 12A) or histidine buffer (FIG. 12B) over a range of temperatures over a 4-week period. [Figure 12B] Representative data are shown showing the relative percentage of intact MC3 in MC3-LNPs encapsulating siRNA formulated in PBS (FIG. 12A) or histidine buffer (FIG. 12B) over a range of temperatures over a 4-week period. [Figure 13A] Representative data are shown demonstrating the stability of encapsulated siRNA sense and antisense strands within MC3-LNP formulated in either PBS (Figure 13A) or histidine buffer (Figure 13B) over a range of temperatures over a 4-week period. [Figure 13B] Representative data are shown demonstrating the stability of encapsulated siRNA sense and antisense strands within MC3-LNP formulated in either PBS (Figure 13A) or histidine buffer (Figure 13B) over a range of temperatures over a 4-week period. [Figure 14] Representative data are shown demonstrating the dose-dependent function of siRNA loaded into MC3-LNPs formulated in either PBS or histidine buffer and stored over a range of temperatures for 4 weeks. [Figure 15] 1 shows that histidine-buffered siRNA-LNPs remained significantly more stable than phosphate-buffered compositions after 3 months of storage at room temperature. [Figure 16A] 1 shows that storage in histidine buffer results in the formation of less siRNA-lipid adducts during storage at various temperatures compared to phosphate buffer compositions. [Figure 16B] Representative data are shown demonstrating that histidine buffer storage inhibits the oxidation of phosphorothioate linkages (PS) to phosphodiester (PO) linkages in chemically modified siRNA. [Figure 17A] Representative data for measurements of mRNA-LNP colloid and payload stability are shown. mRNA-LNP size at 25°C is shown. [Figure 17B] Representative data for the measurement of mRNA-LNP colloid and payload stability are shown. mRNA-LNP polydispersity index (PDI) at 25°C is shown. [Figure 17C] Representative data for measurement of mRNA-LNP colloid and payload stability are shown. mRNA-LNP encapsulation efficiency (EE) at 25°C is shown. [Figure 17D] Representative data for measurements of mRNA-LNP colloid and payload stability are shown. mRNA-LNP RNA content at 25° C. RNA content was observed to decrease in PBS-storage compositions. [Figure 18] Representative data are shown showing that MC3-stabilized mRNA-LNPs experience similar levels of ionic lipid degradation as siRNA-LNPs, and that storage in histidine buffer prevents lipid degradation. [Figure 19] Representative CryoEM images are shown. DETAILED DESCRIPTION OF THE INVENTION

[0026] Aspects of the present disclosure relate to compositions and methods for improving the stability of lipid nanoparticles and one or more active pharmaceutical ingredients (APIs) encapsulated therein. The present disclosure is based in part on compositions comprising components (e.g., histidine buffers at specific pH ranges) that directly or indirectly reduce degradation (e.g., oxidation, hydrolysis, etc.) of one or more lipid components of LNPs. The present disclosure also provides methods for storing the compositions contemplated herein, as well as methods for improving the stability of APIs.

[0027] lipid nanoparticles Aspects of the present disclosure relate to compositions comprising lipid nanoparticles (LNPs). As used herein, the term "LNP" refers to any particle comprising at least one lipid component having an average diameter of less than 1000 nanometers. Exemplary LNPs include, but are not limited to, micelles, liposomes, lipoplexes, and solid lipid nanoparticles, or derivatives thereof. LNPs can have any morphology and structure known in the art. For example, in some embodiments, LNPs are nanospheres, nanorods, nanochains, nanostars, nanoflowers, nanoleafs, nanowhiskers, nanofibers, and nanoboxes. In some embodiments, other morphologies and structures are possible.

[0028] In some embodiments, the LNP comprises a micelle. The micelle can be a micelle or a reverse micelle. In some embodiments, the micelle is an aggregate of amphipathic lipids dispersed in a liquid to form a colloidal suspension. In some embodiments, the amphipathic lipids comprise a hydrophilic head group and a hydrophobic tail group, which, when dispersed in water, spontaneously assemble into a structure that exposes the head group to the aqueous phase and embeds the hydrophobic tail group in the core of the structure. In some embodiments, the lipids are dispersed in an oil phase and spontaneously assemble into a structure that exposes the hydrophobic tail group and embeds the hydrophilic head group (e.g., the lipids form a reverse micelle). In some embodiments, a combination of structures is also possible. For example, in some embodiments, the lipids can be added to an emulsion, such as a water-in-oil emulsion, an oil-in-water emulsion, etc. In some embodiments, lipids added to an oil-in-water emulsion (e.g., water is the continuous phase and oil is the dispersed phase) spontaneously assemble at the water-oil interface with their hydrophilic heads pointing toward the aqueous phase and their hydrophilic tails pointing toward the oil phase. In this configuration, the lipids form a monolayer around the oil droplets, converting them from a hydrophobic to a hydrophilic surface, thus stabilizing the oil droplets in the aqueous phase (e.g., preventing the oil droplets from coalescing). In some embodiments, lipids are added to a water-in-oil emulsion (e.g., oil is the continuous phase and water is the dispersed phase) from a monolayer around water droplets dispersed in the oil phase. In some embodiments, the lipid monolayer can include one or more targeting molecules (e.g., antibodies or fragments thereof, cell-targeting peptides, etc.), drugs, or other agents (e.g., polyethylene glycol). In some embodiments, combinations of emulsions are also possible. For example, in some embodiments, water-in-oil-in-water emulsions are contemplated herein. Such configurations produce stabilized water droplets encapsulated within larger oil droplets. In some embodiments, other combinations are possible, for example, in some embodiments, oil-in-water-in-oil emulsions may be used to make the LNPs disclosed herein.

[0029] In some embodiments, the LNP comprises a liposome. In some embodiments, the liposome is an artificial vesicle having at least one lipid bilayer. In some embodiments, the liposome is a multilamellar vesicle (MLV). In some embodiments, the liposome is a large unilamellar vesicle (LUV). In some embodiments, the liposome is a small unilamellar vesicle (SUV). In some embodiments, the MLV is a large "onion-like" structure comprising several lamellar phase lipid bilayers (e.g., an average diameter of greater than 1000 nanometers). In some embodiments, the LUV comprises a large unilamellar vesicle (e.g., an average diameter between 100 and 200 nanometers). In some embodiments, the SUV comprises a small unilamellar vesicle (e.g., an average diameter of 15 to 30 nanometers). In some embodiments, the lipid bilayer may comprise one or more targeting molecules (e.g., an antibody or fragment thereof, a cell-targeting peptide, etc.), a drug, or other agents (e.g., polyethylene glycol).

[0030] In some embodiments, the LNP comprises a lipoplex. As used herein, the term "lipoplex" refers to a complex formed between a charged liposome (e.g., a cationic or anionic lipid) and at least one oppositely charged component (e.g., a nucleic acid such as an siRNA). As used herein, the at least one oppositely charged component can be a small molecule drug, a polynucleotide (e.g., DNA, RNA, siRNA, miRNA, etc.), a peptide, a polypeptide, a protein (e.g., an antibody), a polymer, or a polysaccharide, comprising at least one cationic (e.g., -NH3+) and / or anionic (e.g., COO-) group. Without wishing to be bound by theory, it is believed that the complex forms due to electrostatic interactions between oppositely charged groups between the charged lipid and the oppositely charged component in the liposome bilayer.

[0031] In some embodiments, any one of the LNPs disclosed herein comprises a solid lipid nanoparticle. In some embodiments, the solid lipid nanoparticle comprises a solid lipid core matrix capable of solubilizing lipophilic molecules (e.g., oils, lipids, charged lipid complexes, DNA, RNA, etc.). In some embodiments, the solid lipid core is stabilized by a lipid monolayer (e.g., similar to a micelle). In some embodiments, the lipid monolayer may comprise one or more targeting molecules (e.g., antibodies or fragments thereof, cell-targeting peptides, etc.), drugs, or other agents (e.g., polyethylene glycol).

[0032] In some embodiments, the LNPs of the disclosure have an average diameter of 1 nanometer or more, 5 nanometers or more, 10 nanometers or more, 50 nanometers or more, 100 nanometers or more, 200 nanometers or more, 300 nanometers or more, 400 nanometers or more, 500 nanometers or more, 600 nanometers or more, 700 nanometers or more, 800 nanometers or more, 900 nanometers or more, or 1000 nanometers or more. In some embodiments, the LNPs have an average diameter of 1000 nanometers or less, 900 nanometers or less, 800 nanometers or less, 700 nanometers or less, 600 nanometers or less, 500 nanometers or less, 400 nanometers or less, 300 nanometers or less, 200 nanometers or less, 100 nanometers or less, 50 nanometers or less, 10 nanometers or less, 5 nanometers or less, or 1 nanometer or less.

[0033] In some embodiments, LNPs have an average diameter of 1 nanometer to 1000 nanometers, 1 nanometer to 350 nanometers, 1 nanometer to 300 nanometers, 1 nanometer to 250 nanometers, 1 nanometer to 200 nanometers, 1 nanometer to 150 nanometers, 1 nanometer to 100 nanometers, or 1 nanometer to 50 nanometers. In some embodiments, the average particle size is 100 nanometers to 900 nanometers, 200 nanometers to 800 nanometers, 300 nanometers to 700 nanometers, or 400 nanometers to 600 nanometers. In some embodiments, LNPs with other average diameters are possible.

[0034] In some embodiments, the LNP comprises one or more lipids (e.g., one or more different types of lipids, e.g., 1, 2, 3, 4, 5, or more different lipids). As used herein, the term lipid refers to any class of organic compounds (e.g., waxes, fats, oils, hormones, lipid membranes, etc.) that are fatty acids or derivatives thereof and are insoluble in water but soluble in organic solvents. In some embodiments, the lipid comprises an amphipathic lipid. In some embodiments, the lipid comprises a neutral lipid. In some embodiments, the lipid comprises one or more ionic lipids. In some embodiments, the one or more ionic lipids comprise a cationic lipid, an anionic lipid, or a combination thereof. In some embodiments, the lipid comprises a functionalized lipid. In some embodiments, the functionalized lipid comprises one or more reactive groups (e.g., an amine, a carboxyl group, etc.). In some embodiments, the lipid comprises a lipid-conjugate, e.g., lipid-polyethylene glycol. In some embodiments, the lipid complex comprises more than one conjugate.

[0035] In some embodiments, the lipid is 1,2-dioleoyl-3-trimethylammonium-propane or a derivative thereof (herein "DOTAP"). In some embodiments, the lipid is 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester or a derivative thereof (e.g., "Dlin-MC3-DMA" or "MC3" herein). In some embodiments, the lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate or a derivative thereof (herein "SM-102"). In some embodiments, the lipid is [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) or a derivative thereof (herein "Alc-0315"). In some embodiments, the lipid is cholesterol or a derivative thereof. In some embodiments, the lipid is (1,2-distearoyl-sn-glycero-3-phosphocholine) or a derivative thereof (herein "DSPC"). In some embodiments, the lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 or a derivative thereof (herein "DMG-PEG-2000").

[0036] In some embodiments, the one or more ionizable lipids comprise MC3 or DOTAP.

[0037] In some embodiments, the compositions of the present disclosure include LNP compositions with varying ratios of one or more lipids. Any suitable ratio known in the art may be used to produce the LNPs disclosed herein. Those skilled in the art will understand that the sum of the ratios from each lipid component cannot exceed 100% (e.g., mol%, wt%, mass%, volume%, etc.). For example, in some embodiments, the percent contribution of each lipid in the LNP composition is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more. In some embodiments, the percent contribution (e.g., mol%) of each lipid in the LNP composition is 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less.

[0038] In some embodiments, the methods include an LNP composition comprising one or more nucleic acids (e.g., siRNA, mRNA, dsRNA, or miRNA). In some embodiments, the concentration of the one or more nucleic acids in the LNP composition is between about 1 ng / mL and 50 mg / mL. In some embodiments, the concentration of the one or more nucleic acids in the LNP composition is between about 1 mg / mL and 50 mg / mL. In some embodiments, the concentration of one or more nucleic acids in the LNP composition is about 500 μg / mL to about 20 mg / mL (e.g., about 500 μg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, or about 20 mg / mL). In some embodiments, the concentration of one or more nucleic acids in the LNP composition is about 500 μg / mL to 3 mg / mL (e.g., about 500 μg / mL, about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, or about 3 mg / mL). In some embodiments, the concentration of one or more nucleic acids in the LNP composition is about 1 mg / mL to about 5 mg / mL, e.g., about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, or about 5 mg / mL. In some embodiments, the concentration of one or more nucleic acids in the LNP composition is about 1.5 mg / mL to 2.5 mg / mL (e.g., about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2.0 mg / mL, about 2.1 mg / mL, about 2.2 mg / mL, about 2.3 mg / mL, about 2.4 mg / mL, or about 2.5 mg / mL).

[0039] In some embodiments, the LNP (e.g., LNP composition) comprises a stabilizer. Without wishing to be bound by theory, the stabilizer is an art-recognized compound that can improve the stability of any one of the LNPs disclosed herein. In some embodiments, the stabilizer comprises a lipid-polyethylene glycol conjugate. In some embodiments, the stabilizer comprises sucrose. Other stabilizers are also possible in some embodiments.

[0040] In some embodiments, the LNPs (e.g., LNP compositions) disclosed herein further comprise an active pharmaceutical ingredient (API). In some embodiments, the LNPs disclosed herein further comprise one or more nucleic acids. In some embodiments, the API comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise RNA. In some embodiments, the RNA is messenger ribonucleic acid (herein "mRNA"), small interfering ribonucleic acid (herein "siRNA"), double-stranded ribonucleic acid (herein "dsRNA"), or micro ribonucleic acid (herein "miRNA").

[0041] In some embodiments, the nucleic acid is siRNA. siRNA is a non-coding double-stranded RNA recognized in the art that operates within the RNA interference pathway. Without being bound by theory, it is believed that they inhibit the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation. In some embodiments, siRNA is a nucleic acid therapeutic agent that targets (e.g., silences or inhibits) genes associated with human diseases or disorders.

[0042] In some embodiments, the one or more nucleic acids are microRNAs (herein "miRNAs"). miRNAs are single-stranded, non-coding RNA molecules recognized in the art to be involved in RNA silencing and post-transcriptional regulation of gene expression. Without being bound by theory, it is believed that miRNAs base-pair with complementary sequences in mRNA, thereby silencing the mRNA molecule through cleavage of the mRNA strand into two fragments or destabilization of the mRNA by shortening its poly(A) tail.

[0043] In some embodiments, one or more nucleic acids are encapsulated within any one of the LNPs disclosed herein. In some embodiments, one or more nucleic acids can be encapsulated within any structure of any one of the LNPs disclosed herein. For example, in some embodiments, one or more nucleic acids can be encapsulated within the hydrophobic lipid bilayer of a liposome or lipoplex. In some embodiments, one or more nucleic acids can be encapsulated within the hydrophilic core of a liposome or lipoplex. In some embodiments, one or more nucleic acids can be encapsulated within the lipophilic core of a solid lipid nanoparticle. In some embodiments, one or more nucleic acids can be encapsulated within one or more reverse micelles that are encapsulated within micelles. In some embodiments, one or more nucleic acids can be electrostatically bound to the outer surface of the LNP. In some embodiments, other configurations are possible.

[0044] Aspects of the present disclosure relate to compositions (e.g., LNPs) comprising a histidine buffer. As used herein, the term "histidine" refers to an essential amino acid comprising an α-amino group, a carboxylic acid group, and an imidazole side chain. In some embodiments, the histidine is L-histidine. In some embodiments, the histidine is D-histidine. In some embodiments, the histidine is a combination of L-histidine and D-histidine.

[0045] In some embodiments, the histidine buffer has a histidine concentration of 5 mM to 50 mM. In some embodiments, the histidine concentration is 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, or 50 mM or more. In some embodiments, the histidine concentration is 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 5 mM or less. In some embodiments, the concentration of histidine in the histidine buffer is 20 mM. In some embodiments, other combinations are possible.

[0046] In some embodiments, the histidine buffer has a histidine concentration of 5 mM to 50 mM, 10 mM to 40 mM, or 20 mM to 30 mM. Other ranges are possible in some embodiments.

[0047] In some embodiments, the histidine buffer has a histidine concentration of 5 mM to 25 mM, 10 mM to 20 mM, 12 mM to 18 mM, or 14 mM to 16 mM. In some embodiments, the histidine buffer has a histidine concentration of at least 5 mM, at least 10 mM, at least 20 mM, at least 25 mM, and at least 30 mM. In some embodiments, the histidine concentration of the histidine buffer is 20 mM.

[0048] In some embodiments, the histidine buffer has a pH value of 5.0 to 7.5 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some embodiments, the histidine buffer has a pH value of about 5.8 to 6.5. In some embodiments, the pH is 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, or 6.5 or higher. In some embodiments, the pH is 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, 5.9 or less, or 5.8 or less.

[0049] In some embodiments, the histidine buffer has a pH of about 5.8 to 6.5. In some embodiments, the pH is 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, or 6.5 or higher. In some embodiments, the pH is 6.5 or lower, 6.4 or lower, 6.3 or lower, 6.2 or lower, 6.1 or lower, 6.0 or lower, 5.9 or lower, or 5.8 or lower.

[0050] In some embodiments, the histidine buffer has a pH of about 5.8 to 6.5. In some embodiments, the histidine buffer has a pH of 5.8 to 6.4, 5.8 to 6.3, 5.8 to 6.2, 5.8 to 6.1, 5.8 to 6.0, or 5.8 to 5.9. In some embodiments, the histidine buffer has a pH of 5.9 to 6.4, 6.0 to 6.3, or 6.1 to 6.2. In some embodiments, the histidine buffer has a pH of 5.9 to 6.4, 6.0 to 6.4, 6.1 to 6.4, 6.2 to 6.4, or 6.3 to 6.4. In some embodiments, the histidine buffer has a pH of about 6.0.

[0051] In some embodiments, the histidine buffer comprises one or more salts. In some embodiments, the histidine buffer comprises sodium chloride. In some embodiments, the concentration of the one or more salts is between 10 mM and 300 mM. In some embodiments, the concentration of the one or more salts is 10 mM or more, 20 mM or more, 40 mM or more, 80 mM or more, 100 mM or more, 120 mM or more, 140 mM or more, 160 mM or more, 180 mM or more, 200 mM or more, 220 mM or more, 240 mM or more, 260 mM or more, 280 mM or more, or 300 mM or more. In some embodiments, the concentration of one or more salts in the histidine buffer is 300 mM or less, 280 mM or less, 260 mM or less, 240 mM or less, 220 mM or less, 200 mM or less, 180 mM or less, 160 mM or less, 140 mM or less, 120 mM or less, 100 mM or less, 80 mM or less, 40 mM or less, 20 mM or less, or 10 mM or less.

[0052] In some embodiments, the concentration of one or more salts is between 10 mM and 300 mM, 20 mM and 280 mM, 40 mM and 260 mM, 80 mM and 240 mM, 100 mM and 220 mM, 120 mM and 200 mM, and 140 mM and 180 mM. In some embodiments, other concentrations are possible, as are other ranges and / or combinations.

[0053] In some embodiments, the histidine buffer contains one or more non-ionic excipients. Examples of non-ionic excipients include sugars (e.g., sucrose), alcohols, polysorbates, etc. In some embodiments, the concentration of the one or more non-ionic excipients is between 10 mM and 300 mM. In some embodiments, the concentration of the one or more non-ionic excipients is 10 mM or more, 20 mM or more, 40 mM or more, 80 mM or more, 100 mM or more, 120 mM or more, 140 mM or more, 160 mM or more, 180 mM or more, 200 mM or more, 220 mM or more, 240 mM or more, 260 mM or more, 280 mM or more, or 300 mM or more. In some embodiments, the concentration of one or more non-ionic excipients in the histidine buffer is 300 mM or less, 280 mM or less, 260 mM or less, 240 mM or less, 220 mM or less, 200 mM or less, 180 mM or less, 160 mM or less, 140 mM or less, 120 mM or less, 100 mM or less, 80 mM or less, 40 mM or less, 20 mM or less, or 10 mM or less.

[0054] A further aspect of the present disclosure relates to the storage of any one of the compositions described herein. For example, in some embodiments, the composition is stored in a container such as a cartridge, a pre-filled syringe, or a vial. In some embodiments, the container is a glass vial. In some embodiments, the container is a polycarbonate vial. All parenteral drugs are stored in Type 1 glass (e.g., USP <660> , EP 3.2.1, ASTM E438) and must meet hydrolysis resistance requirements. However, the composition of glass can vary greatly depending on the manufacturer. Thus, in some embodiments, the vial may be obtained from several different manufacturers. In some embodiments, the vial comprises a cyclic olefin polymer (herein "COP"). In some embodiments, the vial comprises glass. In some embodiments, the glass vial is a Corning Valor® glass vial. In some embodiments, the glass vial is a Schott BT5933 glass vial. In some embodiments, the glass vial is a Gerresheimer BT5974 glass vial. In some embodiments, the glass vial may comprise a coating. Exemplary coatings include, but are not limited to, ammonium sulfate, quartz (e.g., SiOx), SiO2, and the like. In some embodiments, the vial may have a coefficient of expansion (herein "COE") of 33 or 51, although other COEs are contemplated herein. In some embodiments, the vial can have a volume of 1 mL to 20 mL. In some embodiments, the vial has a volume of 1 mL or more, 5 mL or more, 10 mL or more, 15 mL or more, or 20 mL or more. In some embodiments, the vial has a volume of 20 mL or less, 15 mL or less, 10 mL or less, 5 mL or less, or 1 mL or less.

[0055] In some embodiments, the vial includes a pharmaceutical rubber stopper or cap. Any suitable pharmaceutical rubber stopper or cap known in the art can be used herein. In some embodiments, the pharmaceutical rubber stopper or cap is provided with the vial (e.g., Corning Valor® vials are supplied with their own rubber stoppers). In some embodiments, the pharmaceutical rubber stopper is a VS5558 serum stopper.

[0056] In some embodiments, the composition is stored at a temperature above 4° C. In some embodiments, the composition is stored at a temperature of about 5° C. to about 30° C., about 5° C. to about 70° C., about 10° C. to about 60° C., about 15° C. to about 55° C., about 20° C. to about 50° C., about 25° C. to about 45° C., and about 30° C. to 40° C.

[0057] In some embodiments, the composition is not cooled to below 4° C. (eg, the temperature of most commercial refrigerators) or below −20° C. (eg, the temperature of most commercial freezers).

[0058] In some embodiments, the method includes storing the composition at a temperature between 5° C. and 25° C. In some embodiments, the composition is stored at a temperature between 5° C. and 30° C. In some embodiments, the temperature is 5° C. or higher, 10° C. or higher, 15° C. or higher, 20° C. or higher, 25° C. or higher, 30° C. or higher, 35° C. or higher, 40° C. or higher, 45° C. or higher, 50° C. or higher, 55° C. or higher, 60° C. or higher, 65° C. or higher, or 70° C. or higher. In some embodiments, the temperature is 70° C. or lower, 65° C. or lower, 60° C. or lower, 55° C. or lower, 50° C. or lower, 45° C. or lower, 40° C. or lower, 35° C. or lower, 30° C. or lower, 25° C. or lower, 20° C. or lower, 10° C. or lower, or 5° C. or lower. In some embodiments, other combinations are possible (e.g., 5° C. or higher and 30° C. or lower, or 5° C. or higher and 70° C.).

[0059] Aspects of the present disclosure relate to compositions comprising LNPs comprising one or more ionizable lipids and a histidine buffer, as disclosed elsewhere herein. In some embodiments, the one or more ionizable lipids are MC3 or DOTAP. In some embodiments, the histidine buffer has a histidine concentration ranging from about 5 mM to 25 mM. In some embodiments, the histidine buffer has a pH of about 5.8 to about 7.5.

[0060] In some embodiments, compositions comprising histidine-buffered LNP compositions exhibit at least a 200% reduction in the concentration of subvisible particles after 4 weeks of storage at 25°C compared to LNPs stored in phosphate buffer. As used herein, the term "subvisible particles" refers to particles that are too large (e.g., about >0.1 μm) for analysis by size exclusion chromatography (SEC), but small enough (e.g., <100 μm) to be visible to the naked eye. In some embodiments, subvisible particles range in size from about 10 μm to about 25 μm.

[0061] In some embodiments, a composition comprising a histidine-buffered LNP composition exhibits at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 100%, at least a 120%, at least a 140%, at least a 160%, at least a 180%, or at least a 200% reduction in the concentration of subvisible particles compared to LNPs stored in a phosphate buffer after 4 weeks of storage at 25°C.

[0062] In some embodiments, compositions comprising the histidine-buffered LNP composition have an API concentration of at least 0.11 mg / mL or at least 95% encapsulation efficiency compared to LNPs stored in phosphate buffer after 4 weeks of storage at 25° C. In some embodiments, compositions comprising the histidine-buffered LNP composition have an siRNA concentration of at least 0.11 mg / mL or at least 95% encapsulation efficiency compared to LNPs stored in phosphate buffer after 4 weeks of storage at 25° C.

[0063] In some embodiments, a composition comprising a histidine-buffered LNP composition undergoes 0.1% to 2% hydrolysis after 4 weeks of storage at 25° C. In some embodiments, a composition comprising a histidine-buffered LNP composition undergoes 0.1% to 1% oxidation after 4 weeks of storage at 25° C. In some embodiments, a composition comprising a histidine-buffered LNP composition contains 95% to 98.5% intact LNPs after 4 weeks of storage at 25° C.

[0064] method Aspects of the present disclosure relate to one or more methods for producing the LNP compositions disclosed herein. Exemplary LNPs include, but are not limited to, micelles, liposomes, lipoplexes, and solid lipid nanoparticles or derivatives thereof. LNPs can have any morphology and structure known in the art. For example, in some embodiments, LNPs are nanospheres, nanorods, nanochains, nanostars, nanoflowers, nanoleafs, nanowhiskers, nanofibers, and nanoboxes. In some embodiments, other morphologies and structures are possible.

[0065] In some embodiments, the methods include LNP compositions having various ratios of one or more lipids. Any suitable ratio known in the art may be used to produce the LNPs disclosed herein. Those skilled in the art will understand that the sum of the percent contributions from each lipid component cannot exceed 100% (e.g., mol%, wt%, mass%, volume%, etc.). For example, in some embodiments, the percent contribution of each lipid in the LNP composition is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more. In some embodiments, the percent contribution (e.g., mol%) of each lipid in the LNP composition is 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less.

[0066] In some embodiments, the method includes formulating the LNP composition using a histidine buffer. In some embodiments, the histidine buffer has a histidine concentration of 5 mM to 50 mM. In some embodiments, the histidine concentration is 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, or 50 mM or more. In some embodiments, the histidine concentration is 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 5 mM or less. In some embodiments, the concentration of histidine in the histidine buffer is 20 mM. In some embodiments, other combinations are possible.

[0067] In some embodiments, the histidine buffer has a histidine concentration of 5 mM to 25 mM, 10 mM to 20 mM, 12 mM to 18 mM, or 14 mM to 16 mM. In some embodiments, the histidine buffer has a histidine concentration of at least 5 mM, at least 10 mM, at least 20 mM, at least 25 mM, and at least 30 mM. In some embodiments, the histidine concentration of the histidine buffer is 20 mM.

[0068] In some embodiments, the histidine buffer has a histidine concentration of 5 mM to 50 mM, 10 mM to 40 mM, or 20 mM to 30 mM. Other ranges are possible in some embodiments.

[0069] In some embodiments, the histidine buffer has a pH value of 5.0 to 6.5 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 7.5). In some embodiments, the histidine buffer has a pH value of about 5.8 to 6.5. In some embodiments, the pH is 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, or 6.5 or higher. In some embodiments, the pH is 6.5 or lower, 6.4 or lower, 6.3 or lower, 6.2 or lower, 6.1 or lower, 6.0 or lower, 5.9 or lower, or 5.8 or lower.

[0070] In some embodiments, the histidine buffer has a pH of about 5.8 to 6.5. In some embodiments, the pH is 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, or 6.5 or higher. In some embodiments, the pH is 6.5 or lower, 6.4 or lower, 6.3 or lower, 6.2 or lower, 6.1 or lower, 6.0 or lower, 5.9 or lower, or 5.8 or lower.

[0071] In some embodiments, the histidine buffer has a pH of about 5.8 to 6.5. In some embodiments, the histidine buffer has a pH of 5.8 to 6.4, 5.8 to 6.3, 5.8 to 6.2, 5.8 to 6.1, 5.8 to 6.0, or 5.8 to 5.9. In some embodiments, the histidine buffer has a pH of 5.9 to 6.4, 6.0 to 6.3, or 6.1 to 6.2. In some embodiments, the histidine buffer has a pH of 5.9 to 6.4, 6.0 to 6.4, 6.1 to 6.4, 6.2 to 6.4, or 6.3 to 6.4. In some embodiments, the pH of the histidine buffer is 6.0.

[0072] In some embodiments, the histidine buffer comprises one or more salts. In some embodiments, the histidine buffer comprises sodium chloride. In some embodiments, the concentration of the one or more salts is between 10 mM and 300 mM. In some embodiments, the concentration of the one or more salts is 10 mM or more, 20 mM or more, 40 mM or more, 80 mM or more, 100 mM or more, 120 mM or more, 140 mM or more, 160 mM or more, 180 mM or more, 200 mM or more, 220 mM or more, 240 mM or more, 260 mM or more, 280 mM or more, or 300 mM or more. In some embodiments, the concentration of one or more salts in the histidine buffer is 300 mM or less, 280 mM or less, 260 mM or less, 240 mM or less, 220 mM or less, 200 mM or less, 180 mM or less, 160 mM or less, 140 mM or less, 120 mM or less, 100 mM or less, 80 mM or less, 40 mM or less, 20 mM or less, or 10 mM or less.

[0073] In some embodiments, the concentration of one or more salts is between 10 mM and 300 mM, 20 mM and 280 mM, 40 mM and 260 mM, 80 mM and 240 mM, 100 mM and 220 mM, 120 mM and 200 mM, and 140 mM and 180 mM. In some embodiments, other concentrations are possible, as are other ranges and / or combinations.

[0074] Aspects of the present disclosure relate to methods for improving the chemical stability of LNP pharmaceutical compositions, such as those disclosed herein. In some embodiments, the methods include obtaining an LNP pharmaceutical composition comprising a non-histidine buffer. As used herein, the term "non-histidine" buffer refers to any buffer that does not contain the essential amino acid histidine. In some embodiments, the non-histidine buffer comprises a phosphate buffer (e.g., phosphate-buffered saline). In some embodiments, the non-histidine buffer comprises phosphate-buffered saline (PBS). However, other non-histidine buffers are possible according to some embodiments. For example, in some embodiments, the non-histidine buffer is a bicarbonate buffer, a HEPES buffer, a MOPS buffer, a PBST buffer, a TBST buffer, a TE buffer, a TEN buffer, or the like. Any preparation method known in the art can be used to prepare any non-histidine buffer contemplated herein, such as those described in Stoll et al., "Buffers: Principles and practice." Meth. Enzymol. 1990, 182, 24-38.

[0075] In some embodiments, the concentration of the conjugate acid-base pair used to make the non-histidine buffer is between 5 mM and 50 mM. In some embodiments, the concentration is 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, or 50 mM or more. In some embodiments, the concentration is 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, or 5 mM or less. In some embodiments, other combinations are possible.

[0076] In some embodiments, the concentration of the conjugate acid-base pair used to make the non-histidine buffer is 5 mM to 50 mM, 10 mM to 40 mM, or 20 mM to 30 mM, although other ranges are possible in some embodiments.

[0077] In some embodiments, the non-histidine buffer has a pH of 5.8 to 6.5. In some embodiments, the pH is 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, or 6.5 or higher. In some embodiments, the pH is 6.5 or lower, 6.4 or lower, 6.3 or lower, 6.2 or lower, 6.1 or lower, 6.0 or lower, 5.9 or lower, or 5.8 or lower.

[0078] In some embodiments, the non-histidine buffer has a pH value of 5.8 to 6.5. In some embodiments, the non-histidine buffer has a pH value of 5.8 to 6.4, 5.8 to 6.3, 5.8 to 6.2, 5.8 to 6.1, 5.8 to 6.0, or 5.8 to 5.9. In some embodiments, the non-histidine buffer has a pH value of 5.9 to 6.4, 6.0 to 6.3, or 6.1 to 6.2. In some embodiments, the non-histidine buffer has a pH value of 5.9 to 6.4, 6.0 to 6.4, 6.1 to 6.4, 6.2 to 6.4, or 6.3 to 6.4.

[0079] In some embodiments, the non-histidine buffer comprises one or more salts. In some embodiments, the non-histidine buffer comprises sodium chloride. In some embodiments, the concentration of the one or more salts is between 10 mM and 300 mM. In some embodiments, the concentration of the one or more salts is 10 mM or more, 20 mM or more, 40 mM or more, 80 mM or more, 100 mM or more, 120 mM or more, 140 mM or more, 160 mM or more, 180 mM or more, 200 mM or more, 220 mM or more, 240 mM or more, 260 mM or more, 280 mM or more, or 300 mM or more. In some embodiments, the concentration of one or more salts in the non-histidine buffer is 300 mM or less, 280 mM or less, 260 mM or less, 240 mM or less, 220 mM or less, 200 mM or less, 180 mM or less, 160 mM or less, 140 mM or less, 120 mM or less, 100 mM or less, 80 mM or less, 40 mM or less, 20 mM or less, 10 mM or less.

[0080] In some embodiments, the non-histidine buffer contains salt at concentrations of 10 mM to 300 mM, 20 mM to 280 mM, 40 mM to 260 mM, 80 mM to 240 mM, 100 mM to 220 mM, 120 mM to 200 mM, and 140 mM to 180 mM. Other concentrations are possible in some embodiments, as are other ranges and / or combinations.

[0081] In some embodiments, the methods described herein include performing a buffer exchange reaction to replace a non-histidine buffer with a histidine buffer to obtain a histidine-buffered LNP pharmaceutical composition. Any method for exchanging buffers known in the art can be used for the buffer exchange reaction. Exemplary embodiments include, but are not limited to, dialysis, desalting, and diafiltration. In some embodiments, the diafiltration includes performing tangential flow filtration (TFF).

[0082] In some embodiments, the buffer exchange reaction is carried out using dialysis. Without wishing to be bound by theory, dialysis separates small molecules from large molecules by allowing only small molecules to diffuse through a selectively permeable membrane. The solution to be dialyzed (e.g., a histidine-free LNP composition) is placed in a sealed dialysis membrane with a specific molecular weight cutoff and immersed in a selected buffer solution (e.g., a histidine-containing buffer solution). Non-histidine buffer molecules diffuse out of the dialysis bag, and histidine buffer molecules diffuse into the bag (e.g., along their respective diffusion gradients). After the solution reaches equilibrium, the buffer exchange reaction is stopped. To resume the buffer exchange reaction, the dialysate must be replaced with fresh histidine buffer solution to reestablish the concentration gradient. This procedure is repeated until the non-histidine buffer solution is completely removed from the dialysis membrane.

[0083] In some embodiments, the buffer exchange reaction is carried out using desalting. Without wishing to be bound by theory, desalting columns are based on gel filtration chromatography techniques in which a solution containing the buffer to be exchanged (e.g., a non-histidine buffer) is added to a porous resin. Larger molecules (e.g., LNPs) in the solution flow around the porous resin through the voids, while smaller molecules (e.g., non-histidine salts) enter the pores of the porous resin. By passing the sample through a column resin bed of sufficient length and volume, large molecules (e.g., LNPs) can be completely separated from small molecules that migrate longer distances through the pores of the resin bed. It is known in the art that varying the maximum effective pore size (e.g., molecular weight cutoff, also referred to as MWCO) is a major determinant of the size of molecules that can be separated for a particular resin. In some embodiments, any suitable MWCO known in the art may be used to carry out a buffer exchange reaction as disclosed herein.

[0084] In some embodiments, the desalting column can perform a buffer exchange reaction either directly or indirectly. For example, in some embodiments, a desalting column can be used to separate a composition containing LNPs and a non-histidine buffer into LNPs in water and a non-histidine buffer in water. In this case, the desired buffer salt can be added directly to an aqueous solution of LNPs to obtain a final composition containing LNPs in a histidine buffer. Alternatively, the desalting column can be pre-equilibrated with the desired final buffer (e.g., a histidine buffer). In this case, the LNPs mix with the histidine buffer as they pass through the void space and are eluted from the column.

[0085] In some embodiments, desalting can be performed using a variety of formats, such as chromatography columns, gravity flow columns, chromatography cartridges, centrifuge columns, and centrifuge plates. In some embodiments, other formats are possible.

[0086] In some embodiments, the buffer exchange reaction is performed using diafiltration. Without wishing to be bound by theory, diafiltration is a process in which a solution is simultaneously diluted and filtered. In this manner, a composition (e.g., an LNP composition containing a non-histidine buffer) can be filtered to remove small molecules (e.g., salts) while being serially diluted with a desired diluent (e.g., a histidine buffer). As described elsewhere herein, the MWCO of the filter (e.g., resin, dialysis tubing, etc.) plays an important role in determining which salts can be removed and which are retained. Thus, in some embodiments, any suitable MWCO known in the art may be used to perform a buffer exchange reaction as disclosed herein.

[0087] In some embodiments, the method includes storing a histidine-buffered LNP pharmaceutical composition to improve the stability of the composition. Without being bound by theory, it is believed that various lipid components of LNPs may undergo degradation via ester hydrolysis or oxidation of unsaturated bonds when stored in a non-histidine buffer (e.g., phosphate buffer) for extended periods of time at different temperatures. It has now been discovered that storing an LNP pharmaceutical composition in a histidine buffer reduces degradation (e.g., oxidation and / or hydrolysis) of one or more lipid components compared to an LNP pharmaceutical composition stored in a non-histidine buffer (e.g., phosphate buffer).

[0088] In some embodiments, the method includes storing the LNP pharmaceutical composition in a vial. In some embodiments, the composition is stored in a container such as a cartridge, pre-filled syringe, or vial. In some embodiments, the container is a glass vial. In some embodiments, the container is a polycarbonate vial. All parenteral drugs are stored in Type 1 glass (e.g., USP <660> , EP 3.2.1, ASTM E438) and must meet hydrolysis resistance requirements. However, the composition of glass can vary greatly depending on the manufacturer. Thus, in some embodiments, the vial may be obtained from several different manufacturers. In some embodiments, the vial comprises a cyclic olefin polymer (herein "COP"). In some embodiments, the vial comprises glass. In some embodiments, the glass vial is a Corning Valor® glass vial. In some embodiments, the glass vial is a Schott BT5933 glass vial. In some embodiments, the glass vial is a Gerresheimer BT5974 glass vial. In some embodiments, the glass vial may comprise a coating. Exemplary coatings include, but are not limited to, ammonium sulfate, quartz (e.g., SiOx), SiO2, and the like. In some embodiments, the vial may have a coefficient of expansion (herein "COE") of 33 or 51, although other COEs are contemplated herein. In some embodiments, the vial can have a volume of 1 mL to 20 mL. In some embodiments, the vial has a volume of 1 mL or more, 5 mL or more, 10 mL or more, 15 mL or more, or 20 mL or more. In some embodiments, the vial has a volume of 20 mL or less, 15 mL or less, 10 mL or less, 5 mL or less, or 1 mL or less.

[0089] In some embodiments, the vial includes a pharmaceutical rubber stopper or cap. Any suitable pharmaceutical rubber stopper or cap known in the art can be used herein. In some embodiments, the pharmaceutical rubber stopper or cap is provided with the vial (e.g., Corning Valor® vials are supplied with their own rubber stoppers). In some embodiments, the pharmaceutical rubber stopper is a VS5558 serum stopper.

[0090] In some embodiments, the method comprises storing the LNP pharmaceutical composition in a histidine buffer at a temperature above 4° C. In some embodiments, the composition is stored at a temperature between about 5° C. and about 30° C., between about 5° C. and about 70° C., between about 10° C. and about 60° C., between about 15° C. and about 55° C., between about 20° C. and about 50° C., between about 25° C. and about 45° C., and between about 30° C. and 40° C.

[0091] In some embodiments, the composition is not cooled to below 4° C. (e.g., the temperature of most commercially available refrigerators) or below −20° C. (e.g., the temperature of most commercially available freezers). In some embodiments, the composition is frozen (e.g., stored below 0° C., e.g., −20° C. or −70° C.) and then thawed.

[0092] In some embodiments, the method includes storing the composition at a temperature between 5° C. and 25° C. In some embodiments, the composition is stored at a temperature between 5° C. and 30° C. In some embodiments, the temperature is 5° C. or higher, 10° C. or higher, 15° C. or higher, 20° C. or higher, 25° C. or higher, 30° C. or higher, 35° C. or higher, 40° C. or higher, 45° C. or higher, 50° C. or higher, 55° C. or higher, 60° C. or higher, 65° C. or higher, or 70° C. or higher. In some embodiments, the temperature is 70° C. or lower, 65° C. or lower, 60° C. or lower, 55° C. or lower, 50° C. or lower, 45° C. or lower, 40° C. or lower, 35° C. or lower, 30° C. or lower, 25° C. or lower, 20° C. or lower, 10° C. or lower, or 5° C. or lower. In some embodiments, other combinations are possible (e.g., 5° C. or higher and 30° C. or lower, or 5° C. or higher and 70° C.).

[0093] In some embodiments, the method includes adjusting the pH of the histidine buffer during storage to a pH value between 5.8 and 6.5. In some embodiments, the pH is 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, or 6.5 or higher. In some embodiments, the pH is 6.5 or lower, 6.4 or lower, 6.3 or lower, 6.2 or lower, 6.1 or lower, 6.0 or lower, 5.9 or lower, or 5.8 or lower.

[0094] In some embodiments, the method includes adjusting the pH of the histidine buffer during storage to a pH value of 5.8 to 6.5. In some embodiments, the histidine buffer has a pH value of 5.8 to 6.4, 5.8 to 6.3, 5.8 to 6.2, 5.8 to 6.1, 5.8 to 6.0, or 5.8 to 5.9. In some embodiments, the histidine buffer has a pH value of 5.9 to 6.4, 6.0 to 6.3, or 6.1 to 6.2. In some embodiments, the histidine buffer has a pH value of 5.9 to 6.4, 6.0 to 6.4, 6.1 to 6.4, 6.2 to 6.4, or 6.3 to 6.4.

[0095] In some embodiments, the method includes storing the histidine-buffered LNP pharmaceutical composition at 25° C. for 4 weeks, wherein storing the composition increases the number of intact LNPs in the histidine-buffered LNP pharmaceutical composition compared to the LNP pharmaceutical composition stored in a phosphate buffer. For example, in some embodiments, the percent increase in intact LNPs in the histidine-buffered LNP pharmaceutical composition compared to the LNP composition stored in a phosphate buffer is about 0.5% to 14%. In some embodiments, the percent increase is 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, or 14% or more compared to the LNP composition stored in a phosphate buffer after 4 weeks at 25° C. In some embodiments, the percent increase is 14% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less compared to the LNP composition stored in phosphate buffer after 4 weeks at 25°C.

[0096] In some embodiments, the percent increase in intact LNPs in a histidine-buffered LNP pharmaceutical composition compared to an LNP composition stored in a phosphate buffer is between 0.5% and 14%, between 1% and 12%, between 2% and 10%, between 3% and 8%, and between 4% and 6%.

[0097] In some embodiments, the method includes storing a histidine-buffered LNP pharmaceutical composition, wherein storing the composition reduces the percent degradation (e.g., hydrolysis and / or oxidation) of one or more lipid components compared to an LNP composition stored in a phosphate buffer. In some embodiments, the percent reduction in degradation is between 40% and 85% compared to an LNP composition stored in a phosphate buffer. In some embodiments, the percent reduction in degradation is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more compared to an LNP composition stored in a phosphate buffer. In some embodiments, the percent degradation is 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, or 40% or less compared to an LNP composition stored in a phosphate buffer.

[0098] In some embodiments, the method includes storing the histidine-buffered LNP pharmaceutical composition, wherein storing the composition reduces the percent increase in percent hydrolysis by at least 85% after 4 weeks at 25°C compared to an LNP composition stored in phosphate buffer.

[0099] In some embodiments, the percent increase in percent hydrolysis is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% compared to the LNP composition stored in phosphate buffer after 4 weeks at 25°C.

[0100] In some embodiments, the method includes storing a histidine-buffered LNP pharmaceutical composition, wherein storing the composition reduces the percent increase in percent oxidation by at least 33% compared to an LNP composition stored in a phosphate buffer.

[0101] In some embodiments, the percent increase in percent oxidation is reduced by at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 31%, at least 32%, or at least 33% compared to the LNP composition stored in phosphate buffer after 4 weeks at 25°C.

[0102] In some embodiments, the methods disclosed herein increase the colloidal stability of histidine-buffered LNP pharmaceutical compositions compared to LNP pharmaceutical compositions stored in phosphate buffer. Without being bound by theory, it is believed that colloidal instability leads to LNP aggregation, which increases the LNP mean particle size, e.g., as measured using dynamic light scattering, and / or increases the turbidity of the composition, e.g., as measured using transmitted light.

[0103] In some embodiments, storing the histidine-buffered LNP pharmaceutical composition reduces the percent increase in the average particle size of the composition compared to an LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, storing the composition reduces the percent increase in the average particle size by 70% to 100%. In some embodiments, storing the composition reduces the percent increase in the average particle size by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more compared to an LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, storing the composition reduces the percent increase in the average particle size by 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, or 70% or less compared to an LNP composition stored in phosphate buffer after 4 weeks at 25° C.

[0104] In some embodiments, storing the histidine-buffered LNP pharmaceutical composition reduces the percent increase in the average particle size of the composition by about 70% to 100%, about 75% to 95%, or about 80% to 90%.

[0105] In some embodiments, storage of the histidine-buffered LNP pharmaceutical composition results in a 1% to 99% decrease in turbidity of the composition compared to the LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, the percent increase in turbidity of the composition is reduced by 5% or more, 10% or more, 50% or more, 75% or more, or 90% or more compared to the LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, the percent increase in turbidity of the composition is 5% or less, 10% or less, 50% or less, 75% or less, or 90% or less compared to the LNP composition stored in phosphate buffer after 4 weeks at 25° C.

[0106] In some embodiments, storage of the histidine-buffered LNP pharmaceutical composition reduces the turbidity of the composition by about 5% to 10%, about 25% to 75%, or about 50% to 90% after 4 weeks at 25°C compared to an LNP composition stored in phosphate buffer.

[0107] Aspects of the present disclosure further relate to methods for improving the encapsulation efficiency of an API within one or more of the histidine-buffered LNP compositions disclosed herein. Methods for encapsulating APIs (e.g., nucleic acids such as siRNA, miRNA, dsRNA, mRNA, etc.) are generally known, as described, for example, by Mendonca et al., Drug Discov Today. 2023 Mar;28(3):103505.

[0108] In some embodiments, the method includes preparing a first lipid composition in any one of the histidine buffers disclosed herein. Any suitable lipid composition known in the art may be used to generate the first lipid composition. For example, in some embodiments, the first lipid composition includes Dlin-MC3-DMA, cholesterol, DSPC, and DMG-PEG-2k. In some embodiments, the first lipid composition includes SM-102, cholesterol, DSPC, and DMG-PEG-2k. In some embodiments, the first lipid composition includes Alc-0315, cholesterol, DSPC, and DMG-PEG-2k.

[0109] In some embodiments, the lipids in the first lipid composition are mixed in various ratios. Any suitable ratio known in the art can be used to produce the LNPs disclosed herein. Those skilled in the art will understand that the sum of the percent contributions from each lipid component cannot exceed 100% (e.g., mol%, wt%, mass%, volume%, etc.). For example, in some embodiments, the molar percentage of each lipid in the first lipid composition is 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more. In some embodiments, the molar percentage of each lipid in the first lipid composition is 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less.

[0110] In some embodiments, the method comprises preparing a second lipid composition.Any suitable lipid composition known in the art can be used to produce the second lipid composition.In some embodiments, the second lipid composition comprises DOTAP.In some embodiments, the second lipid composition comprises Dlin-MC3-DMA.In some embodiments, other compositions are also possible.

[0111] In some embodiments, the first lipid composition and / or the second lipid composition has a lipid concentration of 10 mM to 15 mM, hi some embodiments, the first lipid concentration and / or the second lipid concentration is 10 mM or more, 10.5 mM or more, 11 mM or more, 11.5 mM or more, 12 mM or more, 12.5 mM or more, 13 mM or more, 13.5 mM or more, 14 mM or more, 14.5 mM or more, or 15 mM or more.

[0112] In some embodiments, the concentration of the first lipid composition and / or the second lipid composition is about 7.5 mM to 17.5 mM, about 8.5 mM to 16.5 mM, about 9.5 mM to 15.5 mM, about 10.5 mM to 14.5 mM, or about 11.5 mM to 13.5 mM.

[0113] In some embodiments, the method includes mixing an API (e.g., mRNA, siRNA, dsRNA, or miRNA) with a second lipid composition. In some embodiments, the API is an siRNA.

[0114] In some embodiments, the method includes mixing one or more nucleic acids with the second lipid composition before mixing the second lipid composition with the first lipid composition. The concentration of the nucleic acid in the second lipid composition can be any concentration that has a therapeutic effect on a subject in need thereof. In some embodiments, the concentration of the one or more nucleic acids in the second lipid composition before mixing the second composition with the first lipid composition is 50 micrograms / mL or more, 75 micrograms / mL or more, 100 micrograms / mL or more, 120 micrograms / mL or more, 150 micrograms / mL or more, 175 micrograms / mL or more, or 200 micrograms / mL or more. In some embodiments, the concentration of one or more nucleic acids in the second lipid composition prior to mixing the second composition with the first lipid composition is 200 micrograms / mL or less, 200 micrograms / mL or less, 175 micrograms / mL or less, 150 micrograms / mL or less, 120 micrograms / mL or less, 100 micrograms / mL or less, 75 micrograms / mL or less, or 50 micrograms / mL or less.

[0115] In some embodiments, the method includes mixing one or more nucleic acids with the second lipid composition in a ratio of 1:50 to 50:1 before mixing the second lipid composition with the first lipid composition. In some embodiments, the one or more nucleic acids are mixed with the second lipid composition in a ratio (e.g., weight percent, molar percent, etc.) of 1:1 or greater, 5:1 or greater, 10:1 or greater, 20:1 or greater, 30:1 or greater, 40:1 or greater, or 50:1 or greater before mixing the first lipid composition with the second lipid composition. In some embodiments, the ratio of the first lipid composition to the second lipid composition is 50:1 or less, 40:1 or less, 30:1 or less, 20:1 or less, 10:1 or less, 5:1 or less, or 1:1 or less (e.g., weight percent, molar percent, etc.) before mixing the first lipid composition with the second lipid composition. In some embodiments, other combinations are possible. In some embodiments, other ranges are also possible.

[0116] In some embodiments, the method includes mixing a first lipid composition with a second lipid composition. The first lipid composition can be added to the second lipid composition in any suitable ratio to form the LNPs disclosed herein. For example, in some embodiments, the ratio of the first lipid composition to the second lipid composition (e.g., 1:1) can be 0.01 to 0.01. st Lipid composition: 2 nd lipid composition) is 1:1 or greater, 0.1:1 or greater, 0.2:1 or greater, 0.3:1 or greater, 0.4:1 or greater, 0.5:1 or greater, 0.5:1 or greater, 0.6:1 or greater, 0.7:1 or greater, 0.8:1 or greater, 0.9:1 or greater, 1:1 or greater, 1:0.9 or greater, 1:0.8 or greater, 1:0.7 or greater, 1:0.6 or greater, 1:0.5 or greater, 1:0.4 or greater, 1:0.3 or greater, 1:0.2 or greater, 1:0.1 or greater. In some embodiments, the ratio of the first lipid composition to the second lipid composition is 1:0.1 or less, 1:0.2 or less, 1:0.3 or less, 1:0.4 or less, 1:0.5 or less, 1:0.6 or less, 1:0.7 or less, 1:0.8 or less, 1:0.9 or less, 1:1 or less, 0.9:1 or less, 0.8:1 or less, 0.7:1 or less, 0.6:1 or less, 0.5:1 or less, 0.4:1 or less, 0.3:1 or less, 0.2:1 or less, or 0.1:1 or less.

[0117] In some embodiments, the ratio of the first lipid composition to the second lipid composition is 1:1 or more, 5:1 or more, 10:1 or more, 20:1 or more, 30:1 or more, 40:1 or more, or 50:1 or more. In some embodiments, the ratio of the first lipid composition to the second lipid composition is 50:1 or less, 40:1 or less, 30:1 or less, 20:1 or less, 10:1 or less, 5:1 or less, or 1:1 or less. In some embodiments, other combinations are possible. In some embodiments, other ranges are possible. In some embodiments, the first lipid composition and the second lipid composition comprise one or more of the same lipids (e.g., MC3, DOTAP, etc.).

[0118] In some embodiments, the methods described herein increase the percent encapsulation efficiency of an API in a histidine-buffered LNP composition compared to an LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, the percent increase in encapsulation efficiency is about 70% to about 140% compared to an LNP composition stored in phosphate buffer after 4 weeks at 25° C.

[0119] In some embodiments, the percent increase in percent encapsulation efficiency of the API in the histidine-buffered LNP composition is between 70% and 140%, between 75% and 135%, between 80% and 130%, between 85% and 125%, between 90% and 120%, between 95% and 110%, or between 100% and 105% compared to an LNP composition stored in phosphate buffer after 4 weeks at 25° C.

[0120] In some embodiments, the percent increase in percent encapsulation efficiency is 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 120% or more, or 140% or more relative to the LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, the percent increase in percent encapsulation efficiency is 140% or less, 120% or less, 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, or 40% or less relative to the LNP composition stored in phosphate buffer after 4 weeks at 25° C.

[0121] In some embodiments, the methods described herein increase the relative percentage of intact ionic lipids in histidine-buffered LNP compositions compared to LNP compositions stored in phosphate buffer after 4 weeks at 25°C. Without wishing to be bound by theory, it is generally believed that mixing negatively charged RNA (e.g., siRNA) with ionic lipids in a second lipid composition (described elsewhere herein) results in the formation of RNA-lipid complexes (e.g., electrostatic bonds). These complexes act to protect the lipids from aqueous buffers, thus reducing the incidence of water-mediated hydrolysis and oxidation.

[0122] In some embodiments, the percent increase in the relative percent of intact ionizable lipids in the histidine-buffered LNP composition is about 55% to about 75% compared to the LNP composition stored in phosphate buffer after 4 weeks at 25°C.

[0123] In some embodiments, the percent increase in the relative percent of intact ionizable lipids in the histidine-buffered LNP composition is about 55% to 75% or about 60% to 70% compared to an LNP composition stored in phosphate buffer after 4 weeks and at 20°C.

[0124] In some embodiments, the percent increase in the relative percent of intact ionizable lipids in the histidine-buffered LNP composition is 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more compared to the LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, the percent increase in the relative percent of intact ionizable lipids in the histidine-buffered LNP composition is 75% or less, 70% or less, 65% or less, 60% or less, or 55% or less compared to the LNP composition stored in phosphate buffer after 4 weeks at 25° C.

[0125] In some embodiments, the methods described herein increase the relative percentage of intact API in a histidine-buffered LNP composition compared to an LNP composition stored in phosphate buffer after 4 weeks at 25° C. In some embodiments, the API comprises an siRNA comprising a sense strand (herein the “SS strand”) and an antisense strand (herein the “AS strand”).

[0126] In some embodiments, the percent increase in the relative percent of intact SS strands in a histidine-buffered LNP composition is at least 100% compared to an LNP composition stored in a phosphate buffer (e.g., if the relative percent of intact SS strands in an LNP composition containing a phosphate buffer is about 40% after 4 weeks at 25°C, and the relative percent of intact SS strands in an LNP composition containing a histidine buffer is about 80% under the same conditions, the percent increase between the two is about 100%.

[0127] In some embodiments, the percent increase in the relative percent of intact SS strands in the histidine-buffered LNP composition is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to an LNP composition stored in phosphate buffer after 4 weeks at 25°C.

[0128] In some embodiments, the percent increase in the relative percent of intact AS strands in the histidine-buffered LNP composition is at least 275% compared to an LNP composition stored in a phosphate buffer (e.g., if the relative percent of intact AS strands in an LNP composition comprising a phosphate buffer is about 20% after 4 weeks at 25°C, and the relative percent of intact SS strands in an LNP composition comprising a histidine buffer is about 75%, then under the same conditions, the percent increase between the two is about 275%.

[0129] In some embodiments, the percent increase in the relative percent of intact AS chains in the histidine-buffered LNP composition is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 250%, or at least 275% compared to an LNP composition stored in phosphate buffer after 4 weeks at 25°C.

[0130] In some embodiments, the methods described herein preserve the biological function of an API encapsulated within a histidine-buffered LNP composition compared to an LNP composition stored in a phosphate buffer after 4 weeks of storage at 25° C. In some embodiments, the methods described herein increase the inhibitory concentration (e.g., IC50) of an siRNA encapsulated within a histidine-buffered LNP composition compared to an LNP composition stored in a phosphate buffer after 4 weeks of storage at 25° C. In some embodiments, the siRNA encapsulated within a histidine-buffered LNP composition exhibits an inhibitory concentration of at least 5 picomolar, at least 10 picomolar, at least 20 picomolar, at least 30 picomolar, at least 40 picomolar, or at least 50 picomolar after 4 weeks of storage at 25° C. In some embodiments, the siRNA encapsulated within a histidine-buffered LNP composition exhibits an inhibitory concentration of at least 50 picomolar. In some embodiments, siRNAs encapsulated within LNP compositions stored in phosphate buffer lack inhibitory concentrations (e.g., they are completely degraded and exhibit no biological activity) after 4 weeks of storage at 25°C.

[0131] Aspects of the present disclosure relate to compositions and methods useful for treating diseases or disorders associated with dysregulated expression of mRNAs and / or their encoded protein products. In some embodiments, the compositions and methods described herein modulate the function, activity, and / or level of a protein product encoded by a target mRNA by decreasing the target mRNA level and / or translation of that target mRNA in a cell or subject. [Example]

[0132] background The stability of the ionic lipids, MC3 and DOTAP (Figure 1), against hydrolytic and oxidative degradation when stored in phosphate buffer was investigated.

[0133] A stock MC3 lipid solution was prepared by dissolving MC3 in ethanol to a final concentration of 4 mg / mL (e.g., 6 mM). Similarly, a stock lipid DOTAP solution was prepared by dissolving DOTAP in ethanol to a final concentration of 4 mg / mL. A stock solution in phosphate-buffered saline (1x, pH 7.4) was used. The final lipid solution was prepared by mixing PBS stock with lipid stock at a 3:1 ratio (e.g., PBS stock: DOTAP stock in ethanol = 3:1; PBS stock: MC3 stock in ethanol = 3:1). Solutions were stored in either glass (BT5933) or polymer (COP) vials.

[0134] The diluted lipid solutions were frozen at -70°C, then thawed and stored at either 5°C, 25°C, or 40°C for either 1 week, 2 weeks, or 4 weeks. At each time point (e.g., after 1 week, 2 weeks, or 4 weeks), the lipid solutions were analyzed by LC-MS to determine the extent of hydrolysis and / or oxidation present.

[0135] As shown in Figure 2A, the percent DOTAP that remained intact (e.g., not degraded) decreased with increasing storage time (e.g., from 1 week to 4 weeks) and was lowest for samples stored at 40°C. Additionally, samples stored in BT5933 glass vials showed better stability when diluted with PBS buffer compared to identical samples stored in COP vials. LC-MS analysis indicated that hydrolysis was the primary degradation pathway for DOTAP (Figure 3A), with oxidation contributing only to degradation at extended storage times and elevated storage temperatures (see Figure 3B, 4 weeks of storage at 40°C).

[0136] Figure 2B shows the percentage (%) of intact MC3 for the various test groups. The % MC3 remaining intact after storage at 5°C and 25°C was lower than the % DOTAP at the same time points, suggesting that MC3 degrades faster than DOTAP under these storage conditions. As noted above, storage in BT5933 glass vials improved stability compared to COP vials when diluted in PBS buffer. LC-MS analysis indicated that the primary degradation pathway for MC3 was oxidation under all conditions tested (see Figure 3C). The data also indicate that hydrolysis is the primary degradation mechanism for DOTAP (Figure 3A).

[0137] Example 1. Study of the chemical stability of lipids in histidine buffer MC3 and DOTAP lipids were prepared using either PBS or histidine buffer. Ethanol stock solutions of MC3 and DOTAP were prepared as described in Example 1. PBS-based solutions were prepared by diluting the stock lipid solution 3:1 (PBS:lipid in ethanol (vol / vol)) with 1x PBS, pH 7.4, respectively. Histidine-based solutions were also prepared by diluting the stock lipid solution 3:1 (histidine:lipid in ethanol vol / vol) with histidine buffer (10 mM, pH 6.0). Solutions were filled in 1 mL aliquots into BT5933, COP, or Valor glass vials to assess stability. An initial control sample was immediately frozen at -70°C.

[0138] The filled vials were stored for either 1 week, 2 weeks, or 4 weeks at either 5° C., 25° C., or 40° C. At each time point (e.g., after 1 week, 2 weeks, or 4 weeks), the lipid solutions were analyzed by LC-MS to determine the extent of hydrolysis and / or oxidation present.

[0139] As shown in Figure 4A, storage in histidine buffer stabilized the % DOTAP intact at all storage conditions tested and also reduced the % hydrolysis of DOTAP compared to storage in PBS, regardless of storage condition (Figure 4B). Furthermore, storage in histidine buffer significantly reduced lipid hydrolysis of MC3 (Figure 5B) and reduced the extent of oxidation (Figure 5C), thus increasing the % intact MC3 at all storage conditions tested (Figure 5A). The data further indicate that storage in Valor glass vials results in a lower oxidation rate for MC3.

[0140] Example 2. Chemical and colloidal stability studies of empty LNPs The stability of empty LNPs containing ionic lipids, MC3 and DOTAP, against hydrolytic and oxidative degradation when stored in either phosphate or histidine buffer was investigated.

[0141] Empty MC3-LNP and DOTAP-LNP were prepared in either PBS (1x, pH 7.4) or histidine buffer (20 mM histidine, 140 mM NaCl, pH 6.0) and stored in either BT5933 (5 mL, 51 COE, Schott) glass vials with VS5558 caps, BT5974 (5 mL, 33 COE, Gerresheimer-treated) glass vials with VS5558 caps, COP vials (5 mL) with matching stoppers, vials with matching caps, or BT5933 Schott vials with VS5558 caps.

[0142] The LNP solutions were stored at either 5° C. or 25° C. for either 0 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, and then frozen at −70° C. At each time point, the LNP solutions were analyzed by LC-MS to determine LNP size (by dynamic light scattering) and degradation by percent hydrolysis and / or oxidation present (by LC-MS).

[0143] As shown in Figure 6A, the composition of the storage buffer had little effect on MC3-LNP size when stored at 5°C. In contrast, Figure 6B shows that histidine buffer improved the colloidal stability of MC3-LNP for up to 4 weeks at room temperature, with the maximum LNP size reaching approximately 80 nm compared to 100-110 nm for the same LNP stored in a PBS-based buffer. Figures 7A and 7B show that the mean particle size of DOTAP-LNP is larger than that of MC3-LNP, regardless of storage conditions.

[0144] Figures 8A and 8B show that the improved size control of MC3-LNPs stored in histidine buffer (Figure 6B) was associated with a slight increase in the percentage of intact MC3-LNPs after a 4-week storage period, a result that was due to a slight decrease in the percent oxidation of MC3 in MC3-LNPs (Figure 8B).

[0145] Figures 9A and 9B show that storage in histidine buffer increased the percentage of intact DOTAP-LNPs after a 4-week storage period at room temperature (Figure 9A), a result that was due to a decrease in the percent hydrolysis of DOTAP in the DOTAP-LNPs (Figure 9B).

[0146] Example 3. Chemical and colloidal stability studies of siRNA-loaded LNPs The stability of siRNA-loaded LNPs containing ionic lipids, MC3 and DOTAP, against hydrolytic and oxidative degradation when stored in either phosphate or histidine buffer was investigated.

[0147] LNPs were prepared using a composition containing Dlin-MC3-DMA, cholesterol, DSPC, and DMG-PEG-2k in a 50:38.5:10:1.5 molar ratio. The composition was mixed with 12.5 mM MC3 or DOTAP lipid, 50 mM sodium citrate (pH 5), and 0.12 ng / mL siRNA (siHPRT) (20:1 wt%). This solution was then passed through an ISCO pump (Teledyne Inc.) and a PD-10 column to obtain the desired LNPs. The LNPs were then diluted 1:1 by volume with either PBS (1x, 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, and 1.8 mM KHPO pH 7.4) or histidine buffer (20 mM histidine, 140 mM NaCl, pH 6.0) to produce the final LNP solution. Both samples were stored in 5933 Schott, 5 mL, 51 COE glass vials.

[0148] The LNP solutions were stored at 5° C. for either 0 days, 2 weeks, or 4 weeks, or at room temperature for either 0 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or 3 months, and then frozen at −70° C. At each time point, the LNP solutions were analyzed via dynamic light scattering (e.g., sizing), polydispersity index (e.g., PDI), encapsulation efficiency, and LC-MS (e.g., stability) to determine the extent of hydrolysis and / or oxidation present.

[0149] Figures 10A and 10B show that storage in histidine buffer produced MC3-LNP with smaller particle size (Figure 10A) and helped maintain a low and relatively constant PDI (Figure 10B). Figures 11A and 11B show that storage in histidine buffer increased encapsulation efficiency, starting at 2 weeks and continuing for up to 4 weeks, at both 5°C and room temperature; storage in histidine buffer also resulted in far fewer subvisible particles with diameters of 2 microns or greater (Figure 11B). Encapsulation efficiency was determined using the Quant-it™ RiboGreen RNA Assay Kit. Figures 12A and 12B show that storage in histidine buffer increased the percentage of intact MC3-LNP to over 95% after a 4-week observation period, regardless of temperature, in contrast to those stored in PBS (approximately 58%, Figure 12A).

[0150] Unexpectedly, storage of siRNA-loaded LNPs in histidine buffer also increased the stability of the encapsulated siRNA compared to those stored in PBS under similar conditions. For example, Figures 13A and 13B show that double-stranded siHPRT degrades over a 4-week storage period when encapsulated in siRNA-LNPs formulated in PBS buffer (Figure 13A). siHPRT encapsulated in LNPs formulated using histidine buffer was compared to room temperature (Figure 13B). After 3 months of storage at room temperature, the histidine-buffered siRNA-LNPs remained significantly more stable than the phosphate-buffered compositions (Figure 15). The properties of the siRNA-loaded LNPs were also evaluated after storage at 40°C. The data show that the histidine-buffered compositions were stable even after 4 weeks of storage at elevated temperatures (Table 1).

[0151] [Table 1]

[0152] Example 4. In vitro knockdown efficiency studies Next, the ability of siRNA-loaded LNPs to knock down genes of interest in vitro was evaluated after storage under a variety of different conditions.

[0153] MC3-LNPs encapsulating siHP at room temperature were prepared as described in Example 3 and stored under one of the conditions shown in Table 1 until needed. To test the effect of storage conditions on transfection efficiency, HeLa cells were added to a 96-well plate (10,000 cells / well), and the appropriate LNP solution (e.g., condition and concentration) or negative control was added to each well. Each group in Table 2 was tested with six different concentrations of RNA in the LNP (e.g., 100 mM, 10 mM, 1 mM, 0.1 mM, 0.01 mM, and 0 mM RNA in the LNP). Thus, each group required six wells per replicate, and each experiment was replicated three times (e.g., each group required 18 wells). Plates were incubated at 37°C and 95 / 5 O2 / CO2 for 24 hours. Subsequently, transfection efficiency was determined by isolating cDNA from each well, and HPRT mRNA concentration was determined by qPCR.

[0154] [Table 2]

[0155] Figure 14 shows that LNPs stored in histidine buffer at room temperature for 4 weeks had IC values ​​similar to freshly prepared LNPs (Group 1) and LNPs formulated in PBS and stored at 4°C for 4 weeks. Thus, LNP solutions formulated in histidine buffer and stored at either 4°C or room temperature maintained the biological function of siHPRT compared to LNPs formulated in PBS and stored at room temperature.

[0156] Figure 16A shows that storage in histidine buffer results in the formation of fewer siRNA-lipid adducts during storage at various temperatures compared to phosphate buffer compositions. Figure 16B shows representative data demonstrating that storage in histidine buffer inhibits the oxidation of phosphorothioate linkages (PS) to phosphodiester (PO) linkages in chemically modified siRNA, resulting in fewer siRNA-lipid adducts.

[0157] Example 5. mRNA-loaded LNPs This example describes the effect of histidine buffering on the colloidal and payload stability of mRNA-loaded LNPs. Briefly, mRNA was formulated at an N / P ratio of 6 using 8 mM Dlin-MC3-DMA and stored in either phosphate buffer (PBS), pH 7.4, or histidine buffer, pH 6.0, at either room temperature or 5°C for 2 or 4 weeks. The compositions were then characterized by MFI, turbidity (by UV), osmolality, DLS, RiboGreen assay, and CryoEM.

[0158] Figures 17A-17D show representative data for measurements of mRNA-LNP colloid and payload stability. Figure 17A shows mRNA-LNP size at 25°C. Figure 17B shows mRNA-LNP polydispersity index (PDI) at 25°C. Figure 17C shows mRNA-LNP encapsulation efficiency (EE) at 25°C. Figure 17D shows the RNA content of mRNA-LNP at 25°C. RNA content was observed to decrease in PBS-storage compositions.

[0159] Figure 18 shows representative data demonstrating that MC3-stabilized mRNA-LNPs experience similar levels of ionic lipid degradation as siRNA-LNPs, and that storage in histidine buffer prevents lipid degradation.

[0160] FIG. 19 shows representative cryo-electron microscopy (CryoEM) images.

Claims

1. (i) a lipid nanoparticle (LNP) comprising one or more ionic lipids; (ii) a histidine buffer having a histidine concentration in the range of 5 mM to 30 mM and a pH in the range of about 5.0 to about 7.

5.

2. 2. The pharmaceutical composition of claim 1, wherein the concentration of histidine buffer ranges from 10 mM to 20 mM histidine.

3. 3. The pharmaceutical composition of claim 1, wherein the concentration of histidine buffer is at least 10 mM.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of histidine buffer is 20 mM.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the pH of the histidine buffer is about 6.

0.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the histidine buffer further comprises one or more salts.

7. 7. The pharmaceutical composition of claim 6, wherein the one or more salts comprise NaCl.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the one or more ionizable lipids comprise an unsaturated tail, and further optionally, the one or more ionizable lipids comprise DLin-MC3-DMA (MC3) and / or dioleoyl-3-trimethylammonium propane (DOTAP).

9. 9. The pharmaceutical composition of claim 8, wherein the one or more ionizable lipids consist of MC3 or DOTAP.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the LNP comprises one or more nucleic acids.

11. The pharmaceutical composition of claim 10 , wherein the one or more nucleic acids comprise RNA.

12. The pharmaceutical composition of claim 11 , wherein the RNA is mRNA, siRNA, dsRNA, or miRNA.

13. The pharmaceutical composition according to claim 11 or 12, wherein the RNA is an siRNA.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the composition is not refrigerated or frozen.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the composition is stored at a temperature above 4°C.

16. 16. The pharmaceutical composition of claim 15, wherein the temperature above 4°C ranges from about 5°C to about 30°C.

17. A container containing the pharmaceutical composition according to any one of claims 1 to 16.

18. 18. The container of claim 17, wherein the container is a pre-filled syringe or a glass vial.

19. 1. A method for improving the chemical stability of a lipid nanoparticle (LNP) pharmaceutical composition, said method comprising: (i) obtaining a non-histidine buffered LNP pharmaceutical composition comprising a non-histidine buffer; (ii) performing a buffer exchange procedure to replace the non-histidine buffer with a histidine buffer having a pH of 5.8 to 7.5 to obtain a histidine-buffered LNP pharmaceutical composition.

20. 20. The method of claim 19, wherein the histidine-buffered LNP pharmaceutical composition comprises one or more ionic lipids, including DLin-MC3-DMA (MC3) and / or dioleoyl-3-trimethylammonium propane (DOTAP).

21. 21. The method of claim 19 or 20, wherein the non-histidine buffer comprises a citrate buffer.

22. 22. The method of claim 21, wherein the pH of the citrate buffer ranges from about 3.5 to about 5.

5.

23. 23. The method of claim 22, wherein the citrate buffer has a pH of 5.

0.

24. The method of any one of claims 19 to 23, wherein the histidine-buffered LNP pharmaceutical composition comprises one or more nucleic acids.

25. 25. The method of claim 24, wherein the one or more nucleic acids comprise RNA.

26. 26. The method of claim 25, wherein the RNA is mRNA, siRNA, dsRNA, or miRNA.

27. 27. The method of claim 25 or 26, wherein the RNA is siRNA.

28. 28. The method of any one of claims 19 to 27, wherein the histidine buffer has a pH of 6.

0.

29. 29. The method of any one of claims 19 to 28, wherein the histidine buffer has a concentration of histidine ranging from about 5 mM to about 30 mM.

30. 30. The method of any one of claims 19 to 29, wherein the histidine buffer has a concentration of histidine ranging from about 10 mM to about 20 mM.

31. 31. The method of claim 29 or 30, wherein the concentration of histidine is at least 10 mM.

32. 31. The method of claim 29 or 30, wherein the concentration of histidine is 20 mM.

33. 33. The method of any one of claims 19-32, wherein the buffer exchange step comprises contacting the non-histidine-buffered LNP pharmaceutical composition with a desalting column.

34. 33. The method of any one of claims 19-32, wherein the buffer exchange procedure comprises contacting the non-histidine-buffered LNP pharmaceutical composition with dialysis tubing or performing tangential flow filtration.

35. 35. The method of any one of claims 19-34, wherein the buffer exchange procedure comprises collecting the histidine-buffered LNP pharmaceutical composition in a container.

36. 36. The method of claim 35, wherein the container is a syringe or a glass vial.

37. 37. The method of any one of claims 19-36, further comprising storing the histidine-buffered LNP pharmaceutical composition at a temperature above 4°C.

38. 38. The method of claim 37, wherein the temperature above 4°C ranges from about 5°C to about 30°C.

39. 39. The method of any one of claims 19-38, wherein the histidine-buffered LNP pharmaceutical composition contains less hydrolyzed lipid compared to a non-histidine-buffered LNP pharmaceutical composition stored in a phosphate buffer.

40. 40. The method of any one of claims 19 to 39, wherein the histidine-buffered LNP pharmaceutical composition contains less oxidized lipids compared to a non-histidine-buffered LNP pharmaceutical composition stored in a phosphate buffer.

41. 41. The method of any one of claims 19-40, wherein the histidine-buffered LNP pharmaceutical composition comprises LNPs with increased colloidal stability compared to non-histidine-buffered LNPs stored in a pharmaceutical composition comprising a phosphate buffer.

42. 1. A method for preparing a lipid nanoparticle (LNP) pharmaceutical composition, said method comprising: (i) preparing a first lipid composition comprising one or more lipids; (ii) preparing a second lipid composition comprising siRNA and an ionic lipid; (iii) mixing the first lipid composition with the second lipid composition; The method, wherein the first lipid composition and the second lipid composition are prepared using a histidine buffer having a pH of 5.8 to 6.5 and a histidine concentration of 5 mM to 30 mM.

43. 1. A method for preparing a lipid nanoparticle (LNP) pharmaceutical composition, said method comprising: (i) obtaining a lipid composition comprising siRNA and an ionic lipid; (ii) mixing the lipid composition with a histidine buffer having a pH of 5.8 to 6.5 and a histidine concentration of 5 mM to 30 mM.

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