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 comprise 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 comprise a histidine buffer. The present disclosure also provides methods for storing compositions of interest herein as well as methods for improving API stability.

Description

[0001] Related Applications

[0002] This application claims the benefit of the filing date of U.S. Provisional Application Serial No. 63 / 435,024, entitled "Stabilization of Lipid Nanoparticle Formulations," filed on December 23, 2022, the entire content of which is incorporated herein by reference. Background of the Invention

[0003] Lipid nanoparticle (LNP) formulations are useful drug delivery systems for encapsulating various active pharmaceutical ingredients (APIs), such as RNAs. LNP formulations typically include an aqueous buffer solution, such as phosphate buffer. However, current LNP formulations are prone to degradation and formation of aggregates during storage at room temperature and thus need improvement.

[0004] Summary of the Invention

[0005] 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 the degradation (e.g., oxidation, hydrolysis, etc.) of one or more lipid components of lipid nanoparticles. In some embodiments, the composition comprises a histidine buffer. In some embodiments, the histidine buffer inhibits the oxidation and / or hydrolysis of one or more lipids of the LNP and / or improves the stability of the API encapsulated within the LNP. The present disclosure also provides methods for storing the compositions of interest herein.

[0006] Thus, in some aspects, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle (LNP) containing one or more ionizable lipids and a histidine buffer having a concentration range of 5 mM to 30 mM of histidine and a pH range of from about 5.0 to about 7.5.

[0007] In some embodiments, the histidine buffer has a concentration range of 10 mM to 20 mM of histidine. In some embodiments, the histidine buffer has a concentration of at least 10 mM. In some embodiments, the histidine buffer has a concentration of 20 mM.

[0008] In some embodiments, the pH of the histidine buffer is about pH 6.0.

[0009] 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 nonionic excipients, such as sucrose.

[0010] In some embodiments, the one or more ionizable lipids comprise unsaturated tails, further optionally wherein the one or more ionizable lipids comprise DLin-MC3-DMA (MC3) and / or dioleoyl-3-trimethylammonium propane (DOTAP). In some embodiments, the one or more ionizable lipids consist of MC3 or DOTAP.

[0011] In some embodiments, the LNP comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids include RNA. In some embodiments, the RNA is mRNA, siRNA, dsRNA, or miRNA. In some embodiments, the RNA is siRNA.

[0012] In some embodiments, the composition is not refrigerated or frozen (e.g., after addition of the histidine buffer). In some embodiments, the composition is stored at a temperature above 4°C. In some embodiments, the temperature range is from about 5°C to about 30°C. In some embodiments, the composition is frozen (e.g., before or after addition of the histidine buffer), then thawed and stored at a temperature in the range of about 5°C to about 30°C.

[0013] In some aspects, the present disclosure provides a container containing the pharmaceutical composition as 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 polymeric vial.

[0014] In some aspects, the present disclosure provides a method 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 operation to replace the non-histidine buffer with a histidine buffer having a pH between 5.8 and 7.5 to obtain a histidine buffered LNP pharmaceutical composition.

[0015] 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.

[0016] In some embodiments, the non-histidine buffer comprises a citrate buffer. In some embodiments, the citrate buffer has a pH range of from about 3.5 to about 5.5. In some embodiments, the citrate buffer has a pH of 5.0.

[0017] In some embodiments, the histidine-buffered LNP pharmaceutical composition comprises one or more nucleic acids. In some embodiments, the one or more nucleic acids include RNA. In some embodiments, the RNA is mRNA, siRNA, dsRNA, or miRNA. In some embodiments, the RNA is siRNA.

[0018] In some embodiments, the histidine buffer has a pH of 6.0. In some embodiments, the histidine buffer has a histidine concentration in the range of about 5 mM to about 30 mM. In some embodiments, the histidine buffer has a histidine concentration in the range of about 10 mM to 20 mM. In some embodiments, the concentration of histidine is at least 10 mM. In some embodiments, the concentration of histidine is 20 mM.

[0019] In some embodiments, the buffer exchange operation comprises contacting the non-histidine-buffered LNP pharmaceutical composition with a desalting column. In some embodiments, the buffer exchange operation comprises contacting the non-histidine-buffered LNP pharmaceutical composition with a dialysis tube 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.

[0020] In some embodiments, the buffer exchange operation comprises collecting the histidine-buffered LNP pharmaceutical composition in a container (such as a vial or 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 polymer vial.

[0021] 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 range is from about 5°C to about 30°C.

[0022] In some embodiments, the histidine-buffered LNP pharmaceutical composition comprises less hydrolyzed lipid as compared to a non-histidine-buffered LNP pharmaceutical composition stored in phosphate buffer.

[0023] In some embodiments, the histidine-buffered LNP pharmaceutical composition comprises less oxidized lipid as compared to a non-histidine-buffered LNP pharmaceutical composition stored in phosphate buffer. In some embodiments, the histidine-buffered LNP pharmaceutical composition comprises LNPs with improved colloidal stability as compared to non-histidine-buffered LNPs stored in a pharmaceutical composition comprising phosphate buffer.

[0024] In some aspects, the present disclosure provides a method for preparing a lipid nanoparticle (LNP) pharmaceutical composition, the method comprising preparing a first lipid composition comprising one or more lipids; preparing a second lipid composition comprising siRNA and an ionizable 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 between 5.8 and 6.5 and a histidine concentration between 5 mM and 30 mM.

[0025] 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 ionizable lipid, and; mixing the lipid composition with a histidine buffer having a pH between 5.8 and 6.5 and a histidine concentration between 5 mM and 30 mM. Brief Description of the Drawings

[0027] Figure 1 Shows the representative structural features of two ionizable lipids, MC3 and DOTAP, used for preparing lipid nanoparticles (LNPs).

[0028] Figure 2A and 2B Show, respectively, the percentages of DOTAP ( Figure 2A ) and MC3 ( Figure 2B ) lipids that remained intact after four weeks of storage at different temperatures in glass (BT5933) or cycloolefin polymer (COP) vials. Briefly, DOTAP or MC3 was dissolved in ethanol at a concentration of 4 mg / mL. Then, the solution was diluted in 1X PBS buffer and filled into glass vials or COP vials.

[0029] Figures 3A - 3C The representative data shown indicate the percentage of hydrolysis ( Figure 3A ) and oxidation ( Figure 3B ) of DOTAP lipid after four weeks of storage at different temperatures in glass (BT5933) or cycloolefin polymer (COP) vials, and the percentage of hydrolysis ( Figure 3C ) of MC3 lipid.

[0030] Figure 4A and 4B The representative data shown indicate that storing DOTAP lipid in a histidine buffer improved the percentage of intact DOTAP lipid after four weeks of storage over a range of temperatures ( Figure 4A ), and that the histidine buffer reduced the hydrolysis of DOTAP lipid during four weeks of storage over a range of temperatures ( Figure 4B)。The histidine buffer had a more significant effect on the chemical stability of DOTAP than the container / seal system.

[0031] Figures 5A - 5C The representative data shown indicate that storing MC3 lipids in histidine buffer improved the percentage of intact MC3 lipids after four weeks of storage over a range of temperatures ( Figure 5A ), and the histidine buffer reduced the hydrolysis ( Figure 5B ) and oxidation ( Figure 5C ) of MC3 lipids during four weeks of storage over a range of temperatures. The histidine buffer had a more significant effect on the chemical stability of MC3 than the container / seal system.

[0032] Figure 6A and 6B The representative data shown indicate that storing empty MC3-LNPs in histidine buffer stabilized the LNP particle size when stored at 5 °C ( Figure 6A ) and room temperature (RT) for four weeks ( Figure 6B ). Histidine buffer was used in the 5933His sample, while 1X PBS buffer was used in the other samples. The BT5933 vial is a type 1 glass vial with a coefficient of expansion (COE) of 51. The BT5974 vial is a type 1 glass vial with a COE of 33.

[0033] Figure 7A and 7B The representative data shown indicate that storing empty DOTAP-LNPs in histidine buffer did not substantially change the LNP particle size compared to PBS buffer when stored at 5 °C or RT for up to four weeks. Histidine buffer was used in the 5933His sample, while 1X PBS buffer was used in the other samples.

[0034] Figure 8A and 8B The representative data shown indicate that storing empty MC3-LNPs in histidine buffer over a range of temperatures for four weeks stabilized the percentage of intact MC3 ( Figure 8A ), and reduced the oxidation of MC3 in the LNPs ( Figure 8B ).

[0035] Figure 9A and 9B The representative data shown indicate that storing empty DOTAP-LNPs in histidine buffer over a range of temperatures for four weeks stabilized the percentage of intact DOTAP ( Figure 9A ), and reduced the hydrolysis of DOTAP in the DOTAP-LNPs ( Figure 9B ).

[0036] Figure 10A and10B Representative data shown indicate the stability of MC3-LNPs loaded with siRNA formulated in histidine buffer compared to formulation in PBS buffer over four weeks across a range of temperatures in terms of particle size ( Figure 10A ) and polydispersity index ( Figure 10B ). All samples were stored in glass vials.

[0037] Figure 11A and 11B Representative data shown indicate that histidine buffer storage stabilized the MC3-LNP encapsulation of siRNA as a function of time over four weeks across a range of temperatures compared to PBS buffer, and reduced the subvisible particle concentration after four weeks across a range of temperatures, as measured by microflow imaging (MFI) ( Figure 11A ). Figure 11B )

[0038] Figure 12A and 12B Representative data shown indicate the relative percentage of intact MC3 in MC3-LNPs encapsulating siRNAs formulated in PBS ( Figure 12A ) or histidine buffer ( Figure 12B ) over four weeks across a range of temperatures.

[0039] Figure 13A and 13B Representative data shown indicate the stability of the sense and antisense strands of siRNA encapsulated within MC3-LNPs formulated in PBS ( Figure 13A ) or histidine buffer ( Figure 13B ) over four weeks across a range of temperatures.

[0040] Figure 14 Representative data shown indicate the dose-dependent function of siRNA loaded within MC3-LNPs formulated in PBS or histidine buffer and stored over four weeks across a range of temperatures.

[0041] Figure 15 Shown is that after three months of storage at room temperature, histidine-buffered siRNA-LNPs remained significantly more stable than phosphate-buffered compositions.

[0042] Figure 16A Shown: Storage in histidine buffer resulted in the formation of fewer siRNA-lipid adducts during storage at various temperatures compared to phosphate-buffered compositions. Figure 16B Representative data shown indicate that histidine buffer storage inhibited the oxidation of phosphorothioate (PS) bonds to phosphodiester (PO) bonds in chemically modified siRNA.

[0043] Figures 17A - 17D Shows representative data for measuring the colloidal stability and payload stability of mRNA-LNPs. Figure 17A Shows the mRNA-LNP size at 25 °C. Figure 17B Shows the polydispersity index (PDI) of mRNA-LNPs at 25 °C. Figure 17C Shows the encapsulation efficiency (EE) of mRNA-LNPs at 25 °C. Figure 17D Shows the mRNA-LNP RNA content at 25 °C. A decrease in RNA content was observed in the compositions stored in PBS.

[0044] Figure 18 The representative data shown indicate that MC3-stabilized mRNA-LNPs undergo a similar level of ionizable lipid degradation as siRNA-LNPs, and storage in histidine buffer prevents lipid degradation.

[0045] Figure 19 Shows representative CryoEM images.

[0046] Detailed description

[0047] 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 in part based on compositions comprising components (such as histidine buffer in certain pH ranges, etc.) that directly or indirectly reduce the degradation (such as oxidation, hydrolysis, etc.) of one or more lipid components of the LNP. The present disclosure also provides methods for storing the compositions of interest herein and methods for improving API stability.

[0048] Lipid nanoparticles

[0049] Aspects of the present disclosure relate to compositions comprising lipid nanoparticles (LNPs). As used herein, the term "LNP" is any particle comprising at least one lipid component with 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, the LNPs are nanospheres, nanorods, nanowires, nanostars, nanoflowers, nanoreefs, nanowhiskers, nanofibers, and nanocapsules. In some embodiments, other morphologies and structures are also possible.

[0050] In some embodiments, the LNP comprises micelles. The micelles can be micelles or reverse micelles. In some embodiments, a micelle is an aggregate of amphiphilic lipids dispersed in a liquid to form a colloidal suspension. In some embodiments, the amphiphilic lipid comprises a hydrophilic head group and a hydrophobic tail group, which spontaneously assemble into a structure with the head group exposed to the aqueous phase and the hydrophobic tail group buried in the structural core when dispersed in water. In some embodiments, the lipid is dispersed into an oil phase and spontaneously assembles into a structure that exposes the hydrophobic tail group and buries the hydrophilic head group (e.g., the lipid forms a reverse micelle). In some embodiments, a combination of structures is also possible. For example, in some embodiments, lipids can be added to an emulsion such as a water-in-oil emulsion, an oil-in-water emulsion, etc. In some embodiments, the 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, where the hydrophilic head points to the aqueous phase and the hydrophilic tail faces the oil phase. In this configuration, the lipids form a monolayer around the oil droplets, converting their hydrophobic surface into a hydrophilic surface, thereby stabilizing the oil droplets in the aqueous phase (e.g., preventing the oil droplets from coalescing). In some embodiments, the lipids added to a water-in-oil emulsion (e.g., oil is the continuous phase and water is the dispersed phase) form a monolayer around the water droplets dispersed in the oil phase. In some embodiments, the lipid monolayer can comprise one or more targeting molecules (e.g., an antibody or a fragment thereof, a cell-targeting peptide, etc.), a drug, or other reagents (e.g., polyethylene glycol). In some embodiments, a combination of emulsions is also possible. For example, in some embodiments, water-in-oil-in-water emulsions are of interest herein. This structure will produce stable water droplets encapsulated within larger oil droplets. In some embodiments, other combinations are also possible. For example, in some embodiments, oil-in-water-in-oil emulsions can be used to produce the LNPs disclosed herein.

[0051] In some embodiments, the LNP comprises liposomes. In some embodiments, a 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, an MLV is a large "onion-like" structure (e.g., with an average diameter greater than 1000 nanometers) comprising several lamellar-phase lipid bilayers. In some embodiments, LUVs comprise large unilamellar vesicles (e.g., with an average diameter between 100 and 200 nanometers). In some embodiments, SUVs comprise small unilamellar vesicles (e.g., with an average diameter between 15 and 30 nanometers). In some embodiments, the lipid bilayer can comprise one or more targeting molecules (e.g., an antibody or a fragment thereof, a cell-targeting peptide, etc.), a drug, or other reagents (e.g., polyethylene glycol).

[0052] In some embodiments, the LNPs comprise lipoplexes. 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 siRNA). As used herein, the at least one oppositely charged component can be a small molecule drug, polynucleotide (e.g., DNA, RNA, siRNA, miRNA, etc.), peptide, polypeptide, protein (e.g., an antibody), polymer, or polysaccharide that contains at least one cation (e.g., -NH 3+ ) and / or anion (e.g., COO - ) group. Without wishing to be bound by theory, it is believed that the complex is formed due to electrostatic interactions between the oppositely charged groups of the charged lipid in the liposome bilayer and the oppositely charged component.

[0053] In some embodiments, any of the LNPs disclosed herein comprise solid lipid nanoparticles. In some embodiments, the solid lipid nanoparticles comprise 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 can comprise one or more targeting molecules (e.g., an antibody or fragment thereof, a cell-targeting peptide, etc.), a drug, or other reagent (e.g., polyethylene glycol).

[0054] In some embodiments, the LNPs of the present disclosure have an average mean diameter greater than or equal to 1 nanometer, greater than or equal to 5 nanometers, greater than or equal to 10 nanometers, greater than or equal to 50 nanometers, greater than or equal to 100 nanometers, greater than or equal to 200 nanometers, greater than or equal to 300 nanometers, greater than or equal to 400 nanometers, greater than or equal to 500 nanometers, greater than or equal to 600 nanometers, greater than or equal to 700 nanometers, greater than or equal to 800 nanometers, greater than or equal to 900 nanometers, greater than or equal to 1000 nanometers. In some embodiments, the LNPs have an average mean diameter less than or equal to 1000 nanometers, less than or equal to 900 nanometers, less than or equal to 800 nanometers, less than or equal to 700 nanometers, less than or equal to 600 nanometers, less than or equal to 500 nanometers, less than or equal to 400 nanometers, less than or equal to 300 nanometers, less than or equal to 200 nanometers, less than or equal to 100 nanometers, less than or equal to 50 nanometers, less than or equal to 10 nanometers, less than or equal to 5 nanometers, less than or equal to 1 nanometer.

[0055] In some embodiments, the LNPs have an average diameter between 1 nanometer and 1000 nanometers, between 1 nanometer and 350 nanometers, between 1 nanometer and 300 nanometers, between 1 nanometer and 250 nanometers, between 1 nanometer and 200 nanometers, between 1 nanometer and 150 nanometers, between 1 nanometer and 100 nanometers, or between 1 nanometer and 50 nanometers. In some embodiments, the average particle size is between 100 nanometers and 900 nanometers, between 200 nanometers and 800 nanometers, between 300 nanometers and 700 nanometers, or between 400 nanometers and 600 nanometers. In some embodiments, LNPs with other average diameters are also possible.

[0056] In some embodiments, the LNPs comprise one or more lipids (e.g., one or more different types of lipids, such as 1, 2, 3, 4, 5, or more different lipids). As used herein, the term lipid refers to any class of organic compounds that are fatty acids or their derivatives and are insoluble in water but soluble in organic solvents (e.g., waxes, fats, oils, hormones, lipid membranes, etc.). In some embodiments, the lipids comprise amphiphilic lipids. In some embodiments, the lipids comprise neutral lipids. In some embodiments, the lipids comprise one or more ionizable lipids. In some embodiments, the one or more ionizable lipids comprise cationic lipids, anionic lipids, or a combination thereof. In some embodiments, the lipids comprise functionalized lipids. In some embodiments, the functionalized lipids comprise one or more reactive groups (e.g., amines, carboxyl groups, etc.). In some embodiments, the lipids comprise lipid-conjugates, such as lipid-polyethylene glycol. In some embodiments, the lipid-conjugates comprise more than one conjugate.

[0057] In some embodiments, the lipid is 1,2-dioleoyl-3-trimethylammonium-propane or a derivative thereof (referred to herein as "DOTAP"). In some embodiments, the lipid is 4-(dimethylamino)-butyric 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" as referred to herein). In some embodiments, the lipid is octadec-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate or a derivative thereof (referred to herein as "SM-102"). In some embodiments, the lipid is [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) or a derivative thereof (referred to herein as "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 (referred to herein as "DSPC"). In some embodiments, the lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 or a derivative thereof (referred to herein as "DMG-PEG-2000").

[0058] In some embodiments, the one or more ionizable lipids comprise MC3 or DOTAP.

[0059] In some embodiments, the compositions of the present disclosure comprise LNP compositions having one or more lipids in different proportions. Any suitable proportions 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 proportions from each lipid component cannot exceed 100% (e.g., mole %, weight %, mass %, volume %, etc.). For example, in some embodiments, the percentage contribution of each lipid in the LNP composition is greater than or equal to 1%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 25%, greater than or equal to 30%, greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 100%. In some embodiments, the percentage contribution (e.g., mole %) of each lipid in the LNP composition is less than or equal to 100%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, less than or equal to 75%, less than or equal to 70%, less than or equal to 65%, less than or equal to 60%, less than or equal to 55%, less than or equal to 50%, less than or equal to 45%, less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 5%, less than or equal to 1%.

[0060] In some embodiments, the method includes an LNP composition comprising one or more nucleic acids (such as 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 the one or more nucleic acids in the LNP composition is between about 500 μg / mL and about 20 mg / mL (such as 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 the one or more nucleic acids in the LNP composition is between about 500 μg / mL and 3 mg / mL (such as 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 the one or more nucleic acids in the LNP composition is between about 1 mg / mL and about 5 mg / mL, such as 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 the one or more nucleic acids in the LNP composition is between about 1.5 mg / mL and 2.5 mg / mL (such as 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).

[0061] In some embodiments, the LNP (such as the LNP composition) comprises a stabilizer. Without wishing to be bound by theory, a stabilizer is a compound recognized in the art that can improve the stability of any of the LNPs disclosed herein. In some embodiments, the stabilizer comprises a lipid-polyethylene glycol conjugate. In some embodiments, the stabilizer comprises sucrose. In some embodiments, other stabilizers are possible.

[0062] 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 include RNA. In some embodiments, the RNA is messenger ribonucleic acid (referred to herein as "mRNA"), small interfering ribonucleic acid (referred to herein as "siRNA"), double-stranded ribonucleic acid (referred to herein as "dsRNA"), or micro ribonucleic acid (referred to herein as "miRNA").

[0063] In some embodiments, the nucleic acid is siRNA. siRNA is a non-coding double-stranded RNA that is well recognized in the art to function within the RNA interference pathway. Without being bound by theory, it is believed that they interfere with the expression of a specific gene having a complementary nucleotide sequence by degrading mRNA post-transcriptionally, thereby preventing translation. In some embodiments, the siRNA is a nucleic acid therapeutic agent that targets (e.g., silences or inhibits) a gene associated with a human disease or disorder.

[0064] In some embodiments, the one or more nucleic acids are microRNA (referred to herein as "miRNA"). miRNA is a single-stranded non-coding RNA molecule that is well 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 mRNAs, which allows them to silence mRNA molecules by cleaving the mRNA strand into two segments or by destabilizing the mRNA by shortening its poly(A) tail.

[0065] In some embodiments, the one or more nucleic acids are encapsulated within any of the LNPs disclosed herein. In some embodiments, the one or more nucleic acids can be encapsulated within any structure of any of the LNPs disclosed herein. For example, in some embodiments, the one or more nucleic acids can be encapsulated within the hydrophobic lipid bilayer of a liposome or lipid complex. In some embodiments, the one or more nucleic acids can be encapsulated within the hydrophilic core of a liposome or lipid complex. In some embodiments, the one or more nucleic acids can be encapsulated within the lipophilic core of a solid lipid nanoparticle. In some embodiments, the one or more nucleic acids can be encapsulated within one or more reverse micelles within a micelle. In some embodiments, the one or more nucleic acids can be electrostatically bound to the outer surface of the LNP. In some embodiments, other structures are possible.

[0066] 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 containing an α-amino group, a carboxylic acid group, and an imidazole side chain. In some embodiments, histidine is L-histidine. In some embodiments, histidine is D-histidine. In some embodiments, histidine is a combination of L-histidine and D-histidine.

[0067] In some embodiments, the histidine buffer has a histidine concentration between 5 mM and 50 mM. In some embodiments, the histidine concentration is greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 30 mM, greater than or equal to 40 mM, or greater than or equal to 50 mM. In some embodiments, the histidine concentration is less than or equal to 50 mM, less than or equal to 40 mM, less than or equal to 30 mM, less than or equal to 20 mM, less than or equal to 10 mM, or less than or equal to 5 mM. In some embodiments, the concentration of histidine in the histidine buffer is 20 mM. In some embodiments, other combinations are also possible.

[0068] In some embodiments, the histidine buffer has a histidine concentration between 5 mM and 50 mM, between 10 mM and 40 mM, or between 20 mM and 30 mM. In some embodiments, other ranges are also possible.

[0069] In some embodiments, the histidine buffer has a histidine concentration between 5 mM and 25 mM, between 10 mM and 20 mM, between 12 mM and 18 mM, or between 14 mM and 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 buffer has a histidine concentration of 20 mM.

[0070] In some embodiments, the histidine buffer has a pH value between 5.0 and 7.5 (such as 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 between 5.8 and 6.5. In some embodiments, the pH is greater than or equal to 5.8, greater than or equal to 5.9, greater than or equal to 6.0, greater than or equal to 6.1, greater than or equal to 6.2, greater than or equal to 6.3, greater than or equal to 6.4, or greater than or equal to 6.5. In some embodiments, the pH is less than or equal to 6.5, less than or equal to 6.4, less than or equal to 6.3, less than or equal to 6.2, less than or equal to 6.1, less than or equal to 6.0, less than or equal to 5.9, or less than or equal to 5.8.

[0071] In some embodiments, the histidine buffer has a pH value between 5.8 and 6.5. In some embodiments, the pH is greater than or equal to 5.8, greater than or equal to 5.9, greater than or equal to 6.0, greater than or equal to 6.1, greater than or equal to 6.2, greater than or equal to 6.3, greater than or equal to 6.4, or greater than or equal to 6.5. In some embodiments, the pH is less than or equal to 6.5, less than or equal to 6.4, less than or equal to 6.3, less than or equal to 6.2, less than or equal to 6.1, less than or equal to 6.0, less than or equal to 5.9, or less than or equal to 5.8.

[0072] In some embodiments, the histidine buffer has a pH value between 5.8 and 6.5. In some embodiments, the histidine buffer has a pH value between 5.8 and 6.4, between 5.8 and 6.3, between 5.8 and 6.2, between 5.8 and 6.1, between 5.8 and 6.0, or between 5.8 and 5.9. In some embodiments, the histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.3, or between 6.1 and 6.2. In some embodiments, the histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.4, between 6.1 and 6.4, between 6.2 and 6.4, or between 6.3 and 6.4. In some embodiments, the histidine buffer has a pH of approximately 6.0.

[0073] 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 greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 40 mM, greater than or equal to 80 mM, greater than or equal to 100 mM, greater than or equal to 120 mM, greater than or equal to 140 mM, greater than or equal to 160 mM, greater than or equal to 180 mM, greater than or equal to 200 mM, greater than or equal to 220 mM, greater than or equal to 240 mM, greater than or equal to 260 mM, greater than or equal to 280 mM, greater than or equal to 300 mM. In some embodiments, the concentration of the one or more salts in the histidine buffer is less than or equal to 300 mM, less than or equal to 280 mM, less than or equal to 260 mM, less than or equal to 240 mM, less than or equal to 220 mM, less than or equal to 200 mM, less than or equal to 180 mM, less than or equal to 160 mM, less than or equal to 140 mM, less than or equal to 120 mM, less than or equal to 100 mM, less than or equal to 80 mM, less than or equal to 40 mM, less than or equal to 20 mM, less than or equal to 10 mM.

[0074] In some embodiments, the concentration of the one or more salts is between 10 mM and 300 mM, between 20 mM and 280 mM, between 40 mM and 260 mM, between 80 mM and 240 mM, between 100 mM and 220 mM, between 120 mM and 200 mM, and between 140 mM and 180 mM. In some embodiments, other concentrations in other ranges and / or combinations are also possible.

[0075] In some embodiments, the histidine buffer comprises one or more non-ionic excipients. Examples of non-ionic excipients include sugars (such as sucrose), alcohols, polysorbates, and the like. 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 greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 40 mM, greater than or equal to 80 mM, greater than or equal to 100 mM, greater than or equal to 120 mM, greater than or equal to 140 mM, greater than or equal to 160 mM, greater than or equal to 180 mM, greater than or equal to 200 mM, greater than or equal to 220 mM, greater than or equal to 240 mM, greater than or equal to 260 mM, greater than or equal to 280 mM, greater than or equal to 300 mM. In some embodiments, the concentration of the one or more non-ionic excipients in the histidine buffer is less than or equal to 300 mM, less than or equal to 280 mM, less than or equal to 260 mM, less than or equal to 240 mM, less than or equal to 220 mM, less than or equal to 200 mM, less than or equal to 180 mM, less than or equal to 160 mM, less than or equal to 140 mM, less than or equal to 120 mM, less than or equal to 100 mM, less than or equal to 80 mM, less than or equal to 40 mM, less than or equal to 20 mM, less than or equal to 10 mM.

[0076] Another aspect of the present disclosure relates to the storage of any of the compositions described herein. For example, in some embodiments, the composition is stored in a container such as a cartridge, a prefilled syringe, or a vial. In some embodiments, the container is a glass vial. In some embodiments, the container is a polycarbonate vial. It is known in the art that all parenteral drugs must be stored in type 1 glass (e.g., USP <660>, EP 3.2.1, ASTM E438) and meet the requirements of hydrolysis resistance. However, the composition of the glass can vary significantly from manufacturer to manufacturer. Thus, in some embodiments, the vials can be obtained from many different manufacturers. In some embodiments, the vial comprises a cyclic olefin polymer (referred to herein as "COP"). In some embodiments, the vial comprises glass. In some embodiments, the glass vial is Corning Glass vials. 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, etc. In some embodiments, the vial may have a coefficient of expansion (referred to herein as "COE") of 33 or 51, but other COEs are also included herein. In some embodiments, the vial may have a volume between 1 ml and 20 ml. In some embodiments, the vial has a volume greater than or equal to 1 mL, greater than or equal to 5 mL, greater than or equal to 10 mL, greater than or equal to 15 mL, or greater than or equal to 20 mL. In some embodiments, the vial has a volume less than or equal to 20 mL, less than or equal to 15 mL, less than or equal to 10 mL, less than or equal to 5 mL or less than or equal to 1 mL.

[0077] In some embodiments, the vial includes a pharmaceutical rubber stopper or cap. Any suitable pharmaceutical rubber stopper or cap known in the art may be used herein. In some embodiments, the pharmaceutical rubber stopper or cap is provided with the vial (e.g., Corning vial is provided with its own rubber stopper). In some embodiments, the pharmaceutical rubber stopper is a VS5558 Serum stopper.

[0078] In some embodiments, the composition is stored 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.

[0079] In some embodiments, the composition is not cooled to less than or equal to 4°C (e.g., the temperature of most commercially available refrigerators) or less than or equal to -20°C (e.g., the temperature of most commercially available freezers).

[0080] 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 greater than or equal to 5°C, greater than or equal to 10°C, greater than or equal to 15°C, greater than or equal to 20°C, greater than or equal to 25°C, greater than or equal to 30°C, greater than or equal to 35°C, greater than or equal to 40°C, greater than or equal to 45°C, greater than or equal to 50°C, greater than or equal to 55°C, greater than or equal to 60°C, greater than or equal to 65°C, or greater than or equal to 70°C. In some embodiments, the temperature is less than or equal to 70°C, less than or equal to 65°C, less than or equal to 60°C, less than or equal to 55°C, less than or equal to 50°C, less than or equal to 45°C, less than or equal to 40°C, less than or equal to 35°C, less than or equal to 30°C, less than or equal to 25°C, less than or equal to 20°C, less than or equal to 10°C or less than or equal to 5°C. In some embodiments, other combinations are possible (e.g., greater than or equal to 5°C and less than or equal to 30°C or greater than or equal to 5°C and less than or equal to 70°C).

[0081] Aspects of the present disclosure relate to compositions comprising LNPs and a histidine buffer disclosed elsewhere herein, the LNPs comprising one or more ionizable lipids. In some embodiments, the one or more ionizable lipids are MC3 or DOTAP. In some embodiments, the histidine buffer has a histidine concentration in the range of about 5 mM to 25 mM. In some embodiments, the histidine buffer has a pH range of about 5.8 to about 7.5.

[0082] In some embodiments, after storage at 25°C for 4 weeks, the concentration of subvisible particles in a composition comprising a histidine-buffered LNP composition is reduced by at least 200% relative to LNPs stored in phosphate buffer. As used herein, the term "subvisible particle" refers to particles that are too large (e.g., ~>0.1 μm) for size exclusion chromatography (SEC) analysis but too small to be visible to the naked eye (e.g., <100 μm). In some embodiments, the size range of subvisible particles is from about 10 μm to about 25 μm.

[0083] In some embodiments, after storage at 25°C for 4 weeks, the concentration of subvisible particles in a composition comprising a histidine-buffered LNP composition is reduced by 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% or at least 200% relative to LNPs stored in phosphate buffer.

[0084] In some embodiments, after storage at 25 °C for 4 weeks, the composition comprising the histidine-buffered LNP composition has an API concentration of at least 0.11 mg / mL or an encapsulation efficiency of at least 95% relative to LNPs stored in phosphate buffer. In some embodiments, after storage at 25 °C for 4 weeks, the composition comprising the histidine-buffered LNP composition has an siRNA concentration of at least 0.11 mg / mL or an encapsulation efficiency of at least 95% relative to LNPs stored in phosphate buffer.

[0085] In some embodiments, after storage at 25 °C for 4 weeks, the composition comprising the histidine-buffered LNP composition undergoes hydrolysis between 0.1% and 2%. In some embodiments, after storage at 25 °C for 4 weeks, the composition comprising the histidine-buffered LNP composition undergoes oxidation between 0.1% and 1%. In some embodiments, after storage at 25 °C for 4 weeks, the composition comprising the histidine-buffered LNP composition contains intact LNPs between 95% and 98.5%.

[0086] Method

[0087] Aspects of the present disclosure relate to one or more methods for preparing the LNP compositions disclosed herein. Exemplary LNPs include, but are not limited to, micelles, liposomes, lipid complexes, and solid lipid nanoparticles or derivatives thereof. LNPs can have any morphology and structure known in the art. For example, in some embodiments, the LNPs are nanospheres, nanorods, nanowires, nanostars, nanoflowers, nanoreefs, nanowhiskers, nanofibers, and nanocapsules. In some embodiments, other morphologies and structures are possible.

[0088] In some embodiments, the method includes an LNP composition having one or more lipids in different proportions. Any suitable proportions 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 percentage contributions from each lipid component cannot exceed 100% (e.g., mole %, weight %, mass %, volume %, etc.). For example, in some embodiments, the percentage contribution of each lipid in the LNP composition is greater than or equal to 1%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 25%, greater than or equal to 30%, greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 100%. In some embodiments, the percentage contribution of each lipid in the LNP composition (e.g., mole %) is less than or equal to 100%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, less than or equal to 75%, less than or equal to 70%, less than or equal to 65%, less than or equal to 60%, less than or equal to 55%, less than or equal to 50%, less than or equal to 45%, less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, or less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 5%, less than or equal to 1%.

[0089] In some embodiments, the method includes formulating the LNP composition using a histidine buffer. In some embodiments, the histidine buffer has a histidine concentration between 5 mM and 50 mM. In some embodiments, the histidine concentration is greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 30 mM, greater than or equal to 40 mM, or greater than or equal to 50 mM. In some embodiments, the histidine concentration is less than or equal to 50 mM, less than or equal to 40 mM, less than or equal to 30 mM, less than or equal to 20 mM, less than or equal to 10 mM, or less than or equal to 5 mM. In some embodiments, the histidine concentration in the histidine buffer is 20 mM. In some embodiments, other combinations are also possible.

[0090] In some embodiments, the histidine buffer has a histidine concentration between 5 mM and 25 mM, between 10 mM and 20 mM, between 12 mM and 18 mM, or between 14 mM and 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 buffer has a histidine concentration of 20 mM.

[0091] In some embodiments, the histidine buffer has a histidine concentration between 5 mM and 50 mM, between 10 mM and 40 mM, or between 20 mM and 30 mM. In some embodiments, other ranges are possible.

[0092] In some embodiments, the histidine buffer has a pH value between 5.0 and 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 between 5.8 and 6.5. In some embodiments, the pH is greater than or equal to 5.8, greater than or equal to 5.9, greater than or equal to 6.0, greater than or equal to 6.1, greater than or equal to 6.2, greater than or equal to 6.3, greater than or equal to 6.4, or greater than or equal to 6.5. In some embodiments, the pH is less than or equal to 6.5, less than or equal to 6.4, less than or equal to 6.3, less than or equal to 6.2, less than or equal to 6.1, less than or equal to 6.0, less than or equal to 5.9, or less than or equal to 5.8.

[0093] In some embodiments, the histidine buffer has a pH value between 5.8 and 6.5. In some embodiments, the pH is greater than or equal to 5.8, greater than or equal to 5.9, greater than or equal to 6.0, greater than or equal to 6.1, greater than or equal to 6.2, greater than or equal to 6.3, greater than or equal to 6.4, or greater than or equal to 6.5. In some embodiments, the pH is less than or equal to 6.5, less than or equal to 6.4, less than or equal to 6.3, less than or equal to 6.2, less than or equal to 6.1, less than or equal to 6.0, less than or equal to 5.9, or less than or equal to 5.8.

[0094] In some embodiments, the histidine buffer has a pH value between 5.8 and 6.5. In some embodiments, the histidine buffer has a pH value between 5.8 and 6.4, between 5.8 and 6.3, between 5.8 and 6.2, between 5.8 and 6.1, between 5.8 and 6.0, or between 5.8 and 5.9. In some embodiments, the histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.3, or between 6.1 and 6.2. In some embodiments, the histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.4, between 6.1 and 6.4, between 6.2 and 6.4, between 6.3 and 6.4. In some embodiments, the histidine buffer has a pH of 6.0.

[0095] 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 greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 40 mM, greater than or equal to 80 mM, greater than or equal to 100 mM, greater than or equal to 120 mM, greater than or equal to 140 mM, greater than or equal to 160 mM, greater than or equal to 180 mM, greater than or equal to 200 mM, greater than or equal to 220 mM, greater than or equal to 240 mM, greater than or equal to 260 mM, greater than or equal to 280 mM, greater than or equal to 300 mM. In some embodiments, the concentration of the one or more salts in the histidine buffer is less than or equal to 300 mM, less than or equal to 280 mM, less than or equal to 260 mM, less than or equal to 240 mM, less than or equal to 220 mM, less than or equal to 200 mM, less than or equal to 180 mM, less than or equal to 160 mM, less than or equal to 140 mM, less than or equal to 120 mM, less than or equal to 100 mM, less than or equal to 80 mM, less than or equal to 40 mM, less than or equal to 20 mM, less than or equal to 10 mM.

[0096] In some embodiments, the concentration of the one or more salts is between 10 mM and 300 mM, between 20 mM and 280 mM, between 40 mM and 260 mM, between 80 mM and 240 mM, between 100 mM and 220 mM, between 120 mM and 200 mM, and between 140 mM and 180 mM. In some embodiments, other concentrations in other ranges and / or combinations are also possible.

[0097] Aspects of the present disclosure relate to methods for improving the chemical stability of LNP pharmaceutical compositions, such as the compositions disclosed herein. In some embodiments, the method comprises 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, according to some embodiments, other non-histidine buffers are possible. For example, in some embodiments, the non-histidine buffer is a bicarbonate buffer, HEPES buffer, MOPS buffer, PBST buffer, TBST buffer, TE buffer, TEN buffer, etc. Any preparation method known in the art can be used to prepare any non-histidine buffer of interest herein, such as those described in Stoll et al., "Buffers: Principles and practice." Meth. Enzymol. 1990. 182, 24–38.

[0098] In some embodiments, the concentration of the conjugate acid-base pair used to generate the non-histidine buffer is between 5 mM and 50 mM. In some embodiments, the concentration is greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 30 mM, greater than or equal to 40 mM, or greater than or equal to 50 mM. In some embodiments, the concentration is less than or equal to 50 mM, less than or equal to 40 mM, less than or equal to 30 mM, less than or equal to 20 mM, less than or equal to 10 mM, or less than or equal to 5 mM. In some embodiments, other combinations are possible.

[0099] In some embodiments, the concentration of the conjugate acid-base pair used to generate the non-histidine buffer is between 5 mM and 50 mM, between 10 mM and 40 mM, or between 20 mM and 30 mM. In some embodiments, other ranges are possible.

[0100] In some embodiments, the non-histidine buffer has a pH value between 5.8 and 6.5. In some embodiments, the pH is greater than or equal to 5.8, greater than or equal to 5.9, greater than or equal to 6.0, greater than or equal to 6.1, greater than or equal to 6.2, greater than or equal to 6.3, greater than or equal to 6.4, or greater than or equal to 6.5. In some embodiments, the pH is less than or equal to 6.5, less than or equal to 6.4, less than or equal to 6.3, less than or equal to 6.2, less than or equal to 6.1, less than or equal to 6.0, less than or equal to 5.9, or less than or equal to 5.8.

[0101] In some embodiments, the non-histidine buffer has a pH value between 5.8 and 6.5. In some embodiments, the non-histidine buffer has a pH value between 5.8 and 6.4, between 5.8 and 6.3, between 5.8 and 6.2, between 5.8 and 6.1, between 5.8 and 6.0, or between 5.8 and 5.9. In some embodiments, the non-histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.3, or between 6.1 and 6.2. In some embodiments, the non-histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.4, between 6.1 and 6.4, between 6.2 and 6.4, or between 6.3 and 6.4.

[0102] 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 greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 40 mM, greater than or equal to 80 mM, greater than or equal to 100 mM, greater than or equal to 120 mM, greater than or equal to 140 mM, greater than or equal to 160 mM, greater than or equal to 180 mM, greater than or equal to 200 mM, greater than or equal to 220 mM, greater than or equal to 240 mM, greater than or equal to 260 mM, greater than or equal to 280 mM, or greater than or equal to 300 mM. In some embodiments, the concentration of the one or more salts in the non-histidine buffer is less than or equal to 300 mM, less than or equal to 280 mM, less than or equal to 260 mM, less than or equal to 240 mM, less than or equal to 220 mM, less than or equal to 200 mM, less than or equal to 180 mM, less than or equal to 160 mM, less than or equal to 140 mM, less than or equal to 120 mM, less than or equal to 100 mM, less than or equal to 80 mM, less than or equal to 40 mM, less than or equal to 20 mM, or less than or equal to 10 mM.

[0103] In some embodiments, the non-histidine buffer comprises salts at concentrations between 10 mM and 300 mM, between 20 mM and 280 mM, between 40 mM and 260 mM, between 80 mM and 240 mM, between 100 mM and 220 mM, between 120 mM and 200 mM, and between 140 mM and 180 mM. In some embodiments, other concentrations in other ranges and / or combinations are also possible.

[0104] In some embodiments, the methods described herein include performing a buffer exchange reaction to replace a non-histidine buffer with a histidine buffer, obtaining a histidine-buffered LNP pharmaceutical composition. The buffer exchange reaction can be performed using any method known in the art for buffer exchange. Exemplary embodiments include, but are not limited to, dialysis, desalting, and diafiltration. In some embodiments, diafiltration includes performing tangential flow filtration (TFF).

[0105] In some embodiments, the buffer exchange reaction is performed 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 selective permeable membrane. The solution to be dialyzed (e.g., an LNP composition without histidine) is placed in a sealed dialysis membrane with a specific molecular weight cut-off value and immersed in a selected buffer (e.g., a buffer containing histidine). Non-histidine buffer molecules diffuse out of the dialysis bag, while histidine buffer molecules diffuse into the bag (e.g., along their respective diffusion gradients). Once the solution reaches equilibrium, the buffer exchange reaction stops. To restart the buffer exchange reaction, the dialysis fluid must be replaced with fresh histidine buffer to re-establish the concentration gradient. The process is repeated until the non-histidine buffer is completely removed from the dialysis membrane.

[0106] In some embodiments, the buffer exchange reaction is performed using desalting. Without wishing to be bound by theory, desalting columns are based on gel filtration chromatography techniques, where a solution containing the buffer to be exchanged (e.g., a non-histidine buffer) is added to a porous resin. Larger molecules in the solution (e.g., LNPs) flow through the porous resin via the void spaces, while smaller molecules (e.g., salts of non-histidine) enter the pores of the porous resin. By passing the sample through a column resin bed of sufficient length and volume, the larger molecules (e.g., LNPs) can be completely separated from the smaller molecules (which travel a longer distance through the pores of the resin bed). It is known in the art that changing the maximum effective pore size is the main determinant of the molecular size that can be separated by a particular resin (e.g., also referred to as the molecular weight cut-off, MWCO). In some embodiments, any suitable MWCO known in the art can be used for the buffer exchange reaction as disclosed herein.

[0107] In some embodiments, the desalting column can perform the buffer exchange reaction directly or indirectly. For example, in some embodiments, the 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 directly added to the aqueous solution of LNPs to obtain the 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, when the LNPs pass through the void spaces and elute from the column, the LNPs are mixed with the histidine buffer.

[0108] In some embodiments, desalting can be performed in a variety of forms, such as chromatography columns, gravity flow columns, chromatography short columns, centrifugal columns, and centrifugal plates. In some embodiments, other forms are also possible.

[0109] In some embodiments, buffer exchange reactions are performed using diafiltration. Without wishing to be bound by theory, diafiltration is the process of simultaneously diluting and filtering a solution. In this way, a composition (such as an LNP composition containing a non-histidine buffer) can be filtered to remove small molecules (such as salts), while being continuously diluted with a desired diluent (such as a histidine buffer). As described elsewhere herein, the MWCO of the filter (such as a resin, dialysis tubing, etc.) plays an important role in determining which salts can be removed and which salts are retained. Thus, in some embodiments, any suitable MWCO known in the art can be used to perform the buffer exchange reactions disclosed herein.

[0110] In some embodiments, the method includes storing a histidine-buffered LNP pharmaceutical composition to improve the stability of the composition. Without wishing to be bound by theory, it is believed that when stored at different temperatures for an extended period of time in a non-histidine buffer (such as a phosphate buffer), the various lipid components of LNPs can undergo degradation via ester hydrolysis or oxidation of unsaturated bonds. It has now been found that storing an LNP pharmaceutical composition in a histidine buffer reduces the degradation (such as oxidation and / or hydrolysis) of one or more lipid components relative to an LNP pharmaceutical composition stored in a non-histidine buffer (such as a phosphate buffer).

[0111] 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. It is known in the art that all parenteral drugs must be stored in type 1 glass (such as USP <660>, EP 3.2.1, ASTM E438) and meet the requirements for hydrolysis resistance. However, the composition of the glass can vary significantly depending on the manufacturer. Thus, in some embodiments, vials can be obtained from a number of different manufacturers. In some embodiments, the vial contains a cycloolefin polymer (referred to herein as "COP"). In some embodiments, the vial contains glass. In some embodiments, the glass vial is Corning Glass vials. 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, etc. In some embodiments, the vial may have a coefficient of expansion (referred to herein as "COE") of 33 or 51, but other COEs are also included herein. In some embodiments, the vial may have a volume between 1 ml and 20 ml. In some embodiments, the vial has a volume greater than or equal to 1 mL, greater than or equal to 5 mL, greater than or equal to 10 mL, greater than or equal to 15 mL, or greater than or equal to 20 mL. In some embodiments, the vial has a volume less than or equal to 20 mL, less than or equal to 15 mL, less than or equal to 10 mL, less than or equal to 5 mL, or less than or equal to 1 mL.

[0112] In some embodiments, the vial comprises a pharmaceutical rubber stopper or cap. Any suitable pharmaceutical rubber stopper or cap known in the art may be used herein. In some embodiments, the pharmaceutical rubber stopper or cap is provided with the vial (e.g., Corning vial is provided with its own rubber stopper). In some embodiments, the pharmaceutical rubber stopper is a VS5558 Serum stopper.

[0113] 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.

[0114] In some embodiments, the composition is not cooled to less than or equal to 4°C (e.g., the temperature of most commercially available refrigerators) or less than or equal to -20°C (e.g., the temperature of most commercially available freezers). In some embodiments, the composition is frozen (e.g., stored below 0°C, such as -20°C or -70°C) and then thawed.

[0115] 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 greater than or equal to 5°C, greater than or equal to 10°C, greater than or equal to 15°C, greater than or equal to 20°C, greater than or equal to 25°C, greater than or equal to 30°C, greater than or equal to 35°C, greater than or equal to 40°C, greater than or equal to 45°C, greater than or equal to 50°C, greater than or equal to 55°C, greater than or equal to 60°C, greater than or equal to 65°C, or greater than or equal to 70°C. In some embodiments, the temperature is less than or equal to 70°C, less than or equal to 65°C, less than or equal to 60°C, less than or equal to 55°C, less than or equal to 50°C, less than or equal to 45°C, less than or equal to 40°C, less than or equal to 35°C, less than or equal to 30°C, less than or equal to 25°C, less than or equal to 20°C, less than or equal to 10°C, or less than or equal to 5°C. In some embodiments, other combinations are possible (e.g., greater than or equal to 5°C and less than or equal to 30°C or greater than or equal to 5°C and less than or equal to 70°C).

[0116] In some embodiments, the method includes adjusting the pH of the histidine buffer to a pH value between 5.8 and 6.5 during storage. In some embodiments, the pH is greater than or equal to 5.8, greater than or equal to 5.9, greater than or equal to 6.0, greater than or equal to 6.1, greater than or equal to 6.2, greater than or equal to 6.3, greater than or equal to 6.4, or greater than or equal to 6.5. In some embodiments, the pH is less than or equal to 6.5, less than or equal to 6.4, less than or equal to 6.3, less than or equal to 6.2, less than or equal to 6.1, less than or equal to 6.0, less than or equal to 5.9, or less than or equal to 5.8.

[0117] In some embodiments, the method includes adjusting the pH of the histidine buffer to a pH value between 5.8 and 6.5 during storage. In some embodiments, the histidine buffer has a pH value between 5.8 and 6.4, between 5.8 and 6.3, between 5.8 and 6.2, between 5.8 and 6.1, between 5.8 and 6.0, or between 5.8 and 5.9. In some embodiments, the histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.3, or between 6.1 and 6.2. In some embodiments, the histidine buffer has a pH value between 5.9 and 6.4, between 6.0 and 6.4, between 6.1 and 6.4, between 6.2 and 6.4, or between 6.3 and 6.4.

[0118] 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 relative to the LNP pharmaceutical composition stored in phosphate buffer. For example, in some embodiments, the percentage increase in intact LNPs in the histidine-buffered LNP pharmaceutical composition is between about 0.5% and 14% relative to the LNP composition stored in phosphate buffer. In some embodiments, after 4 weeks at 25 °C, the percentage increase relative to the LNP composition stored in phosphate buffer is greater than or equal to 0.5%, greater than or equal to 1%, greater than or equal to 2%, greater than or equal to 3%, greater than or equal to 4%, greater than or equal to 5%, greater than or equal to 6%, greater than or equal to 7%, greater than or equal to 8%, greater than or equal to 9%, greater than or equal to 10%, greater than or equal to 11%, greater than or equal to 12%, greater than or equal to 13%, or greater than or equal to 14%. In some embodiments, after 4 weeks at 25 °C, the percentage increase relative to the LNP composition stored in phosphate buffer is less than or equal to 14%, less than or equal to 13%, less than or equal to 12%, less than or equal to 11%, less than or equal to 10%, less than or equal to 9%, less than or equal to 8%, less than or equal to 7%, less than or equal to 6%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%.

[0119] In some embodiments, the percentage increase in intact LNPs in the histidine-buffered LNP pharmaceutical composition is between 0.5% and 14%, between 1% and 12%, between 2% and 10%, between 3% and 8%, and between 4% and 6% relative to the LNP composition stored in phosphate buffer.

[0120] In some embodiments, the method includes storing a histidine-buffered LNP pharmaceutical composition, wherein storing the composition reduces the percentage of degradation (e.g., hydrolysis and / or oxidation) of one or more lipid components relative to an LNP composition stored in phosphate buffer. In some embodiments, the percentage reduction in degradation is between 40% and 85% relative to an LNP composition stored in phosphate buffer. In some embodiments, the percentage reduction in degradation is greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, or greater than or equal to 85% relative to an LNP composition stored in phosphate buffer. In some embodiments, the percentage of degradation is less than or equal to 85%, less than or equal to 80%, less than or equal to 75%, less than or equal to 70%, less than or equal to 65%, less than or equal to 60%, less than or equal to 55%, less than or equal to 50%, less than or equal to 45%, less than or equal to 40% relative to an LNP composition stored in phosphate buffer.

[0121] In some embodiments, the method includes storing a histidine-buffered LNP pharmaceutical composition, wherein after 4 weeks at 25 °C, storing the composition reduces the percentage increase in the percentage of hydrolysis by at least 85% relative to an LNP composition stored in phosphate buffer.

[0122] In some embodiments, after 4 weeks at 25 °C, the percentage increase in the percentage of 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% relative to an LNP composition stored in phosphate buffer.

[0123] In some embodiments, the method includes storing a histidine-buffered LNP pharmaceutical composition, wherein storing the composition reduces the percentage increase in the percentage of oxidation by at least 33% relative to an LNP composition stored in phosphate buffer.

[0124] In some embodiments, after 4 weeks at 25 °C, the percentage increase in the percentage of 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% relative to an LNP composition stored in phosphate buffer.

[0125] In some embodiments, the methods disclosed herein improve the colloidal stability of histidine-buffered LNP drug compositions relative to LNP drug compositions stored in phosphate buffer. Without wishing to be bound by theory, it is believed that colloidal instability results in LNP aggregation, which increases the average LNP particle size (e.g., as measured using dynamic light scattering) and / or increases the turbidity of the composition (e.g., as measured using transmission light).

[0126] In some embodiments, after 4 weeks at 25°C, storage of the histidine-buffered LNP drug composition reduces the percentage increase in the average particle size of the composition relative to an LNP composition stored in phosphate buffer. In some embodiments, storage of the composition reduces the percentage increase in the average particle size by between 70% and 100%. In some embodiments, after 4 weeks at 25°C, storage of the composition reduces the percentage increase in the average particle size relative to an LNP composition stored in phosphate buffer by greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 100%. In some embodiments, after 4 weeks at 25°C, storage of the composition reduces the percentage increase in the average particle size relative to an LNP composition stored in phosphate buffer by less than or equal to 100%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, less than or equal to 75%, or less than or equal to 70%.

[0127] In some embodiments, storage of the histidine-buffered LNP drug composition reduces the percentage increase in the average particle size of the composition by between about 70% and 100%, between about 75% and 95%, or between about 80% and 90%.

[0128] In some embodiments, after 4 weeks at 25°C, storage of the histidine-buffered LNP drug composition reduces the turbidity of the composition by between 1% and 99% relative to an LNP composition stored in phosphate buffer. In some embodiments, after 4 weeks at 25°C, the percentage increase in the turbidity of the composition is reduced by greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 50%, greater than or equal to 75%, or greater than or equal to 90% relative to an LNP composition stored in phosphate buffer. In some embodiments, after 4 weeks at 25°C, the percentage increase in the turbidity of the composition is less than or equal to 5%, less than or equal to 10%, less than or equal to 50%, less than or equal to 75%, or less than or equal to 90% relative to an LNP composition stored in phosphate buffer.

[0129] In some embodiments, after 4 weeks at 25 °C, the histidine-buffered LNP drug composition, relative to the LNP composition stored in phosphate buffer, reduced the turbidity of the composition by between about 5% and 10%, between about 25% and 75%, or between about 50% and 90%.

[0130] Aspects of the present disclosure also relate to methods for improving the encapsulation efficiency of APIs within one or more of the histidine-buffered LNP compositions disclosed herein. Methods for encapsulating APIs (such as nucleic acids like siRNA, miRNA, dsRNA, mRNA, etc.) are generally known, for example, as described by Mendonca et al., Drug Discov Today. March 2023; 28(3):103505.

[0131] In some embodiments, the method includes preparing a first lipid composition in any of the histidine buffers disclosed herein. Any suitable lipid composition known in the art can be used to produce the first lipid composition. For example, in some embodiments, the first lipid composition comprises Dlin-MC3-DMA, cholesterol, DSPC, and DMG-PEG-2k. In some embodiments, the first lipid composition comprises SM-102, cholesterol, DSPC, and DMG-PEG-2K. In some embodiments, the first lipid composition comprises Alc-0315, cholesterol, DSPC, and DMG-PEG-2k.

[0132] 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 prepare the LNPs disclosed herein. Those skilled in the art will understand that the sum of the percentage contributions from each lipid component cannot exceed 100% (e.g., mole %, weight %, mass %, volume %, etc.). For example, in some embodiments, the mole percentage of each lipid in the first lipid composition is greater than or equal to 1%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 25%, greater than or equal to 30%, greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or greater than or equal to 100%. In some embodiments, the mole percentage of each lipid in the first lipid composition is less than or equal to 100%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, less than or equal to 80%, less than or equal to 75%, less than or equal to 70%, less than or equal to 65%, less than or equal to 60%, less than or equal to 55%, less than or equal to 50%, less than or equal to 45%, less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 5%, less than or equal to 1%.

[0133] In some embodiments, the method includes preparing a second lipid composition. Any suitable lipid composition known in the art can be used to prepare 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.

[0134] In some embodiments, the first and / or second lipid composition has a lipid concentration between 10 mM and 15 mM. In some embodiments, the concentration of the first and / or second lipid composition is greater than or equal to 10 mM, greater than or equal to 10.5 mM, greater than or equal to 11 mM, greater than or equal to 11.5 mM, greater than or equal to 12 mM, greater than or equal to 12.5 mM, greater than or equal to 13 mM, greater than or equal to 13.5 mM, greater than or equal to 14 mM, greater than or equal to 14.5 mM, or greater than or equal to 15 mM.

[0135] In some embodiments, the concentration of the first and / or second lipid composition is between about 7.5 mM and 17.5 mM, between about 8.5 mM and 16.5 mM, between about 9.5 mM and 15.5 mM, between about 10.5 mM and about 14.5 mM, or between about 11.5 mM and about 13.5 mM.

[0136] In some embodiments, the method includes mixing an API (such as mRNA, siRNA, dsRNA, or miRNA) with a second lipid composition. In some embodiments, the API is siRNA.

[0137] 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 acids in the second lipid composition can be any concentration that is therapeutically effective for a subject in need thereof. In some embodiments, before mixing the second composition with the first lipid composition, the concentration of the one or more nucleic acids in the second lipid composition is greater than or equal to 50 micrograms / mL, greater than or equal to 75 micrograms / mL, greater than or equal to 100 micrograms / mL, greater than or equal to 120 micrograms / mL, greater than or equal to 150 micrograms / mL, greater than or equal to 175 micrograms / mL, or greater than or equal to 200 micrograms / mL. In some embodiments, before mixing the second composition with the first lipid composition, the concentration of the one or more nucleic acids in the second lipid composition is less than or equal to 200 micrograms / mL, less than or equal to 200 micrograms / mL, less than or equal to 175 micrograms / mL, less than or equal to 150 micrograms / mL, less than or equal to 120 micrograms / mL, less than or equal to 100 micrograms / mL, less than or equal to 75 micrograms / mL, or less than or equal to 50 micrograms / mL.

[0138] In some embodiments, the method includes mixing the one or more nucleic acids with the second lipid composition at a ratio between 1:50 and 50:1 prior to mixing the second lipid composition with the first lipid composition. In some embodiments, prior to mixing the first lipid composition with the second lipid composition, the one or more nucleic acids are mixed with the second lipid composition at a ratio greater than or equal to 1:1, greater than or equal to 5:1, greater than or equal to 10:1, greater than or equal to 20:1, greater than or equal to 30:1, greater than or equal to 40:1, greater than or equal to 50:1 (e.g., weight percentage, mole percentage, etc.). In some embodiments, prior to mixing the first lipid composition with the second lipid composition, the ratio of the first lipid composition to the second lipid composition is less than or equal to 50:1, less than or equal to 40:1, less than or equal to 30:1, less than or equal to 20:1, less than or equal to 10:1, less than or equal to 5:1, or less than or equal to 1:1 (e.g., weight percentage, mole percentage). In some embodiments, other combinations are possible. In some embodiments, other ranges are also possible.

[0139] In some embodiments, the method includes mixing a first lipid composition and 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., first lipid composition:second lipid composition) is greater than or equal to 1:1, greater than or equal to 0.1:1, greater than or equal to 0.2:1, greater than or equal to 0.3:1, greater than or equal to 0.4:1, greater than or equal to 0.5:1, greater than or equal to 0.5:1, greater than or equal to 0.6:1, greater than or equal to 0.7:1, greater than or equal to 0.8:1, greater than or equal to 0.9:1, greater than or equal to 1:1, greater than or equal to 1:0.9, greater than or equal to 1:0.8, greater than or equal to 1:0.7, greater than or equal to 1:0.6, greater than or equal to 1:0.5, greater than or equal to 1:0.4, greater than or equal to 1:0.3, greater than or equal to 1:0.2, greater than or equal to 1:0.1. In some embodiments, the ratio of the first lipid composition to the second lipid composition is less than or equal to 1:0.1, less than or equal to 1:0.2, less than or equal to 1:0.3, less than or equal to 1:0.4, less than or equal to 1:0.5, less than or equal to 1:0.6, less than or equal to 1:0.7, less than or equal to 1:0.8, less than or equal to 1:0.9, less than or equal to 1:1, less than or equal to 0.9:1, less than or equal to 0.8:1, less than or equal to 0.7:1, less than or equal to 0.6:1, less than or equal to 0.5:1, less than or equal to 0.4:1, less than or equal to 0.3:1, less than or equal to 0.2:1, less than or equal to 0.1:1.

[0140] In some embodiments, the ratio of the first lipid composition to the second lipid composition is greater than or equal to 1:1, greater than or equal to 5:1, greater than or equal to 10:1, greater than or equal to 20:1, greater than or equal to 30:1, greater than or equal to 40:1, greater than or equal to 50:1. In some embodiments, the ratio of the first lipid composition to the second lipid composition is less than or equal to 50:1, less than or equal to 40:1, less than or equal to 30:1, less than or equal to 20:1, less than or equal to 10:1, less than or equal to 5:1, or less than or equal to 1:1. 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.).

[0141] In some embodiments, after 4 weeks at 25°C, the method described herein increases the percentage of API encapsulation within the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer. In some embodiments, after 4 weeks at 25°C, the percentage increase in encapsulation is between about 70% and about 140% relative to the LNP composition stored in phosphate buffer.

[0142] In some embodiments, after 4 weeks at 25°C, the percentage increase in the percentage of API encapsulation within the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer 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%.

[0143] In some embodiments, after 4 weeks at 25°C, the percentage increase in encapsulation percentage is greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 100%, greater than or equal to 120%, or greater than or equal to 140% relative to the LNP composition stored in phosphate buffer. In some embodiments, after 4 weeks at 25°C, the percentage increase in encapsulation percentage is less than or equal to 140%, less than or equal to 120%, less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, less than or equal to 50%, or less than or equal to 40% relative to the LNP composition stored in phosphate buffer.

[0144] In some embodiments, after 4 weeks at 25 °C, the methods described herein increase the relative percentage of intact ionizable lipid within the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer. Without wishing to be bound by theory, it is generally believed that mixing negatively charged RNAs (such as siRNA) with ionizable lipids in a second lipid composition (described elsewhere herein) results in the formation of RNA-lipid complexes (such as electrostatic bonds). These complexes serve to protect the lipids from the aqueous buffer, thereby reducing the incidence of water-mediated hydrolysis and oxidation.

[0145] In some embodiments, after 4 weeks at 25 °C, the percentage increase in the relative percentage of intact ionizable lipid in the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer is between about 55% and about 75%.

[0146] In some embodiments, after 4 weeks at 20 °C, the percentage increase in the relative percentage of intact ionizable lipid in the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer is between about 55% and about 75% or between about 60% and 70%.

[0147] In some embodiments, after 4 weeks at 25 °C, the percentage increase in the relative percentage of intact ionizable lipid in the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer is greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, or greater than or equal to 75%. In some embodiments, after 4 weeks at 25 °C, the percentage increase in the relative percentage of intact ionizable lipid in the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer is less than or equal to 75%, less than or equal to 70%, less than or equal to 65%, less than or equal to 60%, or less than or equal to 55%.

[0148] In some embodiments, after 4 weeks at 25 °C, the methods described herein increase the relative percentage of intact APIs within the histidine-buffered LNP composition relative to the LNP composition stored in phosphate buffer. In some embodiments, the API comprises siRNA, and the siRNA comprises a sense strand (the "SS-strand" herein) and an antisense strand (the "AS-strand" herein).

[0149] In some embodiments, the percentage increase in the relative percentage of intact SS-chains in the histidine-buffered LNP composition, relative to the LNP composition stored in phosphate buffer, is at least 100% (e.g., if the relative percentage of intact SS-chains in the LNP composition containing phosphate buffer is ~40% after 4 weeks at 25°C and the relative percentage of intact SS-chains in the LNP composition containing histidine buffer is ~80% under the same conditions, then the percentage increase between the two is approximately ~100%).

[0150] In some embodiments, after 4 weeks at 25°C, the percentage increase in the relative percentage of intact SS-chains in the histidine-buffered LNP composition, relative to the LNP composition stored in phosphate buffer, 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%.

[0151] In some embodiments, the percentage increase in the relative percentage of intact AS-chains in the histidine-buffered LNP composition, relative to the LNP composition stored in phosphate buffer, is at least 275% (e.g., if the relative percentage of intact AS-chains in the LNP composition containing phosphate buffer is ~20% after 4 weeks at 25°C and the relative percentage of intact AS-chains in the LNP composition containing histidine buffer is ~75% under the same conditions, then the percentage increase between the two is approximately ~275%).

[0152] In some embodiments, after 4 weeks at 25°C, the percentage increase in the relative percentage of intact AS-chains in the histidine-buffered LNP composition, relative to the LNP composition stored in phosphate buffer, 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%.

[0153] In some embodiments, after 4 weeks at 25 °C, the methods described herein preserve the biological function of APIs encapsulated within histidine-buffered LNP compositions relative to LNP compositions stored in phosphate buffer. In some embodiments, after 4 weeks at 25 °C, the methods described herein increase the inhibitory concentration (e.g., IC50) of siRNAs encapsulated within histidine-buffered LNP compositions relative to LNP compositions stored in phosphate buffer. In some embodiments, after 4 weeks at 25 °C, siRNAs encapsulated within histidine-buffered LNP compositions exhibit an inhibitory concentration of at least 5 pM (picomolar), at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, or at least 50 pM. In some embodiments, siRNAs encapsulated within histidine-buffered LNP compositions exhibit an inhibitory concentration of at least 50 pM. In some embodiments, after 4 weeks at 25 °C, siRNAs encapsulated within LNP compositions stored in phosphate buffer lack an inhibitory concentration (e.g., they are completely degraded and do not exhibit biological activity).

[0154] Aspects of the present disclosure relate to compositions and methods useful for treating diseases or disorders associated with dysregulated expression of an mRNA and / or its encoded protein product. 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 reducing the level of the target mRNA and / or translation of the target mRNA in a cell or subject being treated. Examples

[0155] Background

[0156] The stability of ionizable lipids MC3 and DOTAP ( Figure 1 ) to hydrolysis and oxidative degradation was investigated when stored in phosphate buffer.

[0157] A stock MC3 lipid solution was prepared by dissolving MC3 in an ethanol solution to a final concentration of 4 mg / mL (e.g., 6 mM). Similarly, a stock lipid DOTAP solution was prepared by dissolving DOTAP in an ethanol solution to a final concentration of 4 mg / mL. A stock solution of phosphate-buffered saline (1x, pH 7.4) was used. Final lipid solutions were prepared by mixing the PBS stock solution with the lipid stock solution in a 3:1 ratio (e.g., PBS stock solution:DOTAP ethanol stock solution = 3:1; PBS stock solution:MC3 ethanol stock solution = 3:1). The solutions were stored in glass (BT5933) or polymer (COP) vials.

[0158] The diluted lipid solution was frozen at -70 °C and then thawed, and stored at 5 °C, 25 °C or 40 °C for 1 week, 2 weeks or 4 weeks. At each time point (e.g., after 1 week, 2 weeks or 4 weeks), the lipid solution was analyzed by LC-MS to determine the extent of hydrolysis and / or oxidation present.

[0159] As Figure 2A shown, the percentage of DOTAP remaining intact (e.g., undegraded) decreased with increasing storage time (e.g., 1 week > 4 weeks), and the samples stored at 40 °C were the lowest. Also, when diluted with PBS buffer, the samples stored in BT5933 glass vials showed better stability compared to the same samples stored in COP vials. LC-MS analysis indicated that the main degradation pathway of DOTAP was hydrolysis ( Figure 3A ), while oxidation only contributed to degradation at extended storage times and elevated storage temperatures ( Figure 3B , see storage at 40 °C for 4 weeks).

[0160] Figure 2B The percentage of intact MC3 (%) for each test group is shown. The percentage of MC3 remaining intact after storage at 5 °C and 25 °C was lower than the percentage of DOTAP at the same time points, indicating that MC3 degraded faster than DOTAP under these storage conditions. As described above, when diluted in PBS buffer, storage in BT5933 glass vials improved stability relative to COP vials. LC-MS analysis indicated that the main degradation pathway of MC3 was oxidation under all test conditions (see Figure 3C ). The data indicate that hydrolysis is the main degradation mechanism for DOTAP ( Figure 3A ).

[0161] Example 1. Study on the chemical stability of lipids in histidine buffer

[0162] MC3 and DOTAP lipids were prepared using 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 solutions with 1x PBS (pH 7.4) at 3:1 (PBS:lipid in ethanol (volume / volume)). Histidine-based solutions were also prepared by diluting the lipid stock solutions with histidine buffer (10 mM, pH 6.0) at 3:1 (histidine:lipid in ethanol, volume / volume). The solutions were filled into 1 mL aliquots in BT5933, COP or Valor glass vials and equilibrated. Initial control samples were immediately frozen at -70 °C.

[0163] Store the filled vials at 5 °C, 25 °C, or 40 °C for 1 week, 2 weeks, or 4 weeks. At each time point (e.g., after 1 week, 2 weeks, or 4 weeks), analyze the lipid solution by LC-MS to determine the extent of hydrolysis and / or oxidation present.

[0164] As Figure 4A shown, at all tested storage conditions, the intact DOTAP% was stabilized in histidine buffer and the hydrolysis% of DOTAP was reduced relative to storage in PBS, regardless of the storage conditions ( Figure 4B ). Additionally, storage in histidine buffer significantly reduced the lipid hydrolysis of MC3 ( Figure 5B ) and decreased the extent of oxidation ( Figure 5C ), thus increasing the intact MC3% at all tested storage conditions ( Figure 5A ). The data further showed that storage in Valor vials resulted in a lower oxidation rate of MC3.

[0165] Example 2. Study on the chemical and colloidal stability of empty LNP

[0166] The stability of empty LNPs containing the ionizable lipids MC3 and DOTAP against hydrolysis and oxidative degradation was investigated when stored in phosphate buffer or histidine buffer.

[0167] Empty MC3-LNPs and DOTAP-LNPs were prepared in PBS (1x, pH 7.4) or histidine buffer (20 mM histidine, 140 mM NaCl, pH 6.0) and stored in 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, Valor vials with matching caps, or BT5933 Schott vials with VS5558 caps.

[0168] Store the LNP solution at 5 °C or 25 °C for 0 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, then freeze at -70 °C. At each time point, analyze the LNP solution by LC-MS to determine the LNP size (by dynamic light scattering) and the degradation via hydrolysis and / or oxidation present (by LC-MS).

[0169] As Figure 6A shown, when stored at 5 °C, the composition of the storage buffer had little effect on the MC3-LNP size. In contrast, Figure 6BIt was shown that at room temperature, histidine buffer improved the colloidal stability of MC3-LNPs for up to 4 weeks, with the maximum LNP size reaching approximately 80 nm, compared to 100 - 110 nm for the same LNPs stored in PBS-based buffer. Figure 7A and 7B It was shown that regardless of the storage conditions, the average particle size of DOTAP-LNPs was larger than that of MC3-LNPs.

[0170] Figure 8A and 8B It was shown that the improvement in 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, which was due to a slight decrease in the percentage of oxidized MC3 in MC3-LNPs ( Figure 8B ).

[0171] Figure 9A and 9B It was shown that after a 4-week storage period at room temperature, storing in histidine buffer increased the percentage of intact DOTAP-LNPs ( Figure 9A ), which was due to a decrease in the percentage of hydrolyzed DOTAP in DOTAP-LNPs ( Figure 9B ).

[0172] Example 3. Study on the chemical and colloidal stability of siRNA - loaded LNP

[0173] The stability of siRNA-loaded LNPs containing the ionizable lipids MC3 and DOTAP against hydrolysis and oxidative degradation was investigated when stored in phosphate buffer or histidine buffer.

[0174] LNPs were prepared using a composition containing Dlin-MC3-DMA, cholesterol, DSPC, DMG-PEG-2k in a molar ratio of 50:38.5:10:1.5. The composition was mixed with MC3 or DOTAP lipid at a concentration of 12.5 mM, 50 mM sodium citrate (pH 5), and siRNA (siHPRT) at a concentration of 0.12 ng / mL (20:1 weight %). Then, the solution was passed through an ISCO pump (Teledyne Inc.) and a PD-10 column to obtain the desired LNPs. Subsequently, the LNPs were diluted 1:1 in volume with PBS (1x, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) or histidine buffer (20 mM histidine, 140 mM NaCl, pH 6.0) to obtain the final LNP solution. Both samples were stored in 5933 Schott (5 mL, 51 COE) glass vials.

[0175] Store the LNP solution at 5 °C for 0 days, 2 weeks, or 4 weeks; or at room temperature for 0 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or 3 months, then freeze at -70 °C. At each time point, analyze the LNP solution by dynamic light scattering (e.g., size determination), polydispersity index (e.g., PDI), encapsulation efficiency, and LC-MS (e.g., stability) to determine the extent of hydrolysis and / or oxidation present.

[0176] Figure 10A and 10B showed that storage in histidine buffer produced MC3-LNPs with smaller particle sizes ( Figure 10A ), and helped maintain a low and relatively constant PDI ( Figure 10B ). Figure 11A and 11B showed that storage in histidine buffer increased the encapsulation efficiency starting at 2 weeks and continuing through 4 weeks at 5 °C and room temperature; storage in histidine buffer also produced far fewer subvisible particles with diameters greater than or equal to 2 microns ( Figure 11B ). Encapsulation efficiency was determined by the Quant-it TM RiboGreen RNA Assay Kit. Figure 12A and 12B showed that after a 4-week observation period, storage in histidine buffer increased the percentage of intact MC3-LNPs ( Figure 12B to over 95%, as opposed to those stored in PBS (~58%, Figure 12A ).

[0177] Surprisingly, relative to those stored in PBS under similar conditions, LNPs loaded with siRNA stored in histidine buffer also improved the stability of the encapsulated siRNAs. For example, Figure 13A and 13B showed that when encapsulated within siRNA-LNPs formulated in PBS buffer, double-stranded siHPRT degraded over a 4-week storage period ( Figure 13A ); compared to siHPRT encapsulated in LNPs formulated with histidine buffer ( Figure 13B ). After storage at room temperature for 3 months, histidine-buffered siRNA-LNPs remained significantly more stable than phosphate-buffered compositions ( Figure 15 ). The properties of siRNA-loaded LNPs were also evaluated after storage at 40 °C. The data showed that even after storage at elevated temperature for 4 weeks, the histidine-buffered compositions were stable (Table 1).

[0178] Table 1

[0179]

[0180]

[0181] Example 4. Study on in vitro knockdown efficiency

[0182] Next, the ability of siRNA-loaded LNPs to knockdown genes of interest in vitro after storage under various different conditions was evaluated.

[0183] MC3-LNPs encapsulating siHPRT 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 an appropriate LNP solution (e.g., conditions and concentrations) or negative control was added to each well. Each group in Table 2 was tested at 6 different concentrations of RNA in the LNPs (e.g., 100 mM, 10 mM, 1 mM, 0.1 mM, 0.01 mM, and 0 mM of RNA in the LNP). Thus, each group required 6 wells per repeat and each experiment was repeated 3 times (e.g., each group required 18 wells). The 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 measuring the concentration of HPRT mRNA by qPCR.

[0184] Table 2. Conditions for intracellular studies

[0185]

[0186] Figure 14 Shown: LNPs stored in histidine buffer for 4 weeks at room temperature had IC50 values similar to freshly prepared LNPs (Group 1) and LNPs formulated in PBS and stored at 4 °C for 4 weeks. Thus, relative to LNPs formulated in PBS and stored at room temperature, LNP solutions formulated in histidine buffer and stored at 4 °C or room temperature maintained the biological function of siHPRT.

[0187] Figure 16A Shown: Storage in histidine buffer resulted in the formation of fewer siRNA-lipid adducts during storage at various temperatures relative to phosphate-buffered compositions. Figure 16B Representative data shown indicate that storage in histidine buffer inhibited the oxidation of phosphorothioate (PS) bonds to phosphodiester (PO) bonds in chemically modified siRNA.

[0188] Example 5. mRNA - loaded LNPs

[0189] This example describes the effect of histidine buffer pairs on the stability of mRNA-loaded LNP colloids and payloads. Briefly, mRNAs were formulated with 8 mM DLin-MC3-DMA at an N / P ratio of 6 and stored in phosphate buffer (PBS) at pH 7.4 or histidine buffer at pH 6.0 at room temperature or 5 °C for 2 or 4 weeks. The compositions were then characterized by MFI, turbidity (by UV), osmotic pressure, DLS, RiboGreen assay, and CryoEM.

[0190] Figures 17A - 17D Representative data for measuring the stability of mRNA-LNP colloids and payloads are shown. Figure 17A The mRNA-LNP size at 25 °C is shown. Figure 17B The mRNA-LNP polydispersity index (PDI) at 25 °C is shown. Figure 17C The mRNA-LNP encapsulation efficiency (EE) at 25 °C is shown. Figure 17D The mRNA-LNP RNA content at 25 °C is shown. A decrease in RNA content was observed in the PBS-stored compositions.

[0191] Figure 18 The representative data shown indicate that MC3-stabilized mRNA-LNPs undergo a similar level of ionizable lipid degradation as siRNA-LNPs, and storage in histidine buffer prevents lipid degradation.

[0192] Figure 19 Representative cryo-electron microscopy (CryoEM) images are shown.

Claims

1. A pharmaceutical composition comprising: (i) a lipid nanoparticle (LNP) comprising one or more ionizable lipids; and (ii) a histidine buffer having a concentration of 5 mM to 30 mM histidine and a pH of about 5.0 to about 7.

5.

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

3. The pharmaceutical composition of claim 1 or 2, wherein the concentration of the histidine buffer is at least 10 mM.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the concentration of the 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 pH 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. The pharmaceutical composition of claim 6, wherein the one or more salts comprise NaCl.

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

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 of claim 11 or 12, wherein the RNA is 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. The pharmaceutical composition of claim 15, wherein the temperature above 4°C is about 5°C to about 30°C.

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

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

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

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

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

22. The method of claim 21, wherein the citrate buffer has a pH in the range of about 3.5 to about 5.

5.

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

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

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

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

0.

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

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

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

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

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

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

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

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

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

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

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

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

41. The method of any one of claims 19 to 40, wherein the histidine-buffered LNP pharmaceutical composition contains LNPs with improved colloidal stability relative to the non-histidine-buffered LNPs stored in a pharmaceutical composition containing phosphate buffer.

42. A method for preparing a lipid nanoparticle (LNP) pharmaceutical composition, the method comprising: (i) preparing a first lipid composition comprising one or more lipids; (ii) preparing a second lipid composition comprising siRNA and an ionizable lipid, and (iii) 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 between 5.8 and 6.5 and a histidine concentration between 5 mM and 30 mM.

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

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