Method for detecting particles using centrifugal field-flow fractionation

Abstract

To provide a method for detecting lipid nanoparticles.SOLUTION: The present disclosure provides a method for detecting size, density, or both size and density of lipid nanoparticles. The detection can be performed using centrifugal field flow fractionation with a carrier solution containing monosaccharides.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure includes a method for separating particles by centrifugal field-flow fractionation. [Background technology]

[0002] Nanomedicine involves the use of nanotechnology to produce nanoparticles of active pharmaceutical ingredients or to formulate nanoparticles for use in drug delivery, etc. Accurately understanding the relationship between the physicochemical properties of nanoparticles, such as particle size, morphology, and surface properties, and the corresponding biological responses is an important challenge. Particle size and particle size distribution are considered to be important quality attributes of nanomedicines.

[0003] Currently, electron microscopy and dynamic light scattering (DLS) are used to characterize the size and shape of nanoparticles. DLS, in particular, is commonly used to investigate particle size distributions in the field of nanomedicine. Field-flow fractionation (FFF), which uses various fields, including asymmetric flow, centrifugal, electric, thermal, and magnetic, has recently attracted considerable attention for determining the precise size distribution of nanoparticles. Among FFF techniques, asymmetric flow field-flow fractionation (AF4) offers high resolution by classifying nanoparticles based on their diffusion coefficients (i.e., hydrodynamic size) in the presence of particles of different sizes in a polydisperse mixture. AF4 is increasingly being used to investigate the size, stability, and drug release potential of nanomedicines. In contrast, centrifugal FFF (CF3), also known as sedimentation field-flow fractionation, can separate particles based on their size and the density difference between the particles and the elution fluid. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent Application Publication No. 2020 / 0001505 Summary of the Invention [Problem to be solved by the invention]

[0005] The physicochemical properties of nanoparticles used as nanomedicines have not been evaluated based on particle size and density. If the particle size and density of nanoparticles can be evaluated, it may be possible to easily perform advanced evaluations of their quality and physicochemical properties. [Means for solving the problem]

[0006] In one aspect, disclosed herein is a method for detecting, profiling, sorting, and isolating nanoparticles based on the particle size and density of nanoparticles used in nanomedicines using CF3. The nanoparticles can be lipid nanoparticles. Also disclosed herein is a method for administering lipid nanoparticles isolated and prepared using CF3. Also disclosed herein is a pharmaceutical composition comprising lipid nanoparticles containing a therapeutic agent and sorted and / or isolated using CF3.

[0007] In some embodiments, a method for sorting lipid nanoparticles may include sorting lipid nanoparticles based on size, density, or size and density by CF3 using a carrier solution containing a simple sugar. The simple sugar may be glucose. In some embodiments, the lipid nanoparticles may include a therapeutic agent, such as a messenger RNA molecule. The messenger RNA molecule may encode a vaccine. In other embodiments, the therapeutic agent encapsulated in the lipid nanoparticles may be an influenza vaccine, a cancer drug, or an RNAi drug. In some embodiments, the carrier solution may be a phosphate buffer.

[0008] According to some embodiments, a method for detecting lipid nanoparticles may include sorting lipid nanoparticles based on size, density, or size and density by CF3 using a carrier solution containing a monosaccharide, and detecting lipid nanoparticles containing a therapeutic agent. In some embodiments, the detecting step may be performed using at least one detector selected from the group consisting of a multi-angle light scattering (MALS) detector, a photodiode array detector, and an absorbance detector. The monosaccharide may be glucose. In some embodiments, the lipid nanoparticles may contain a therapeutic agent, such as a messenger RNA molecule. The messenger RNA molecule may encode a vaccine. In other embodiments, the therapeutic agent encapsulated in the lipid nanoparticles may be an influenza vaccine, a cancer drug, or an RNAi drug. In some embodiments, the carrier solution may be a phosphate buffer. The method may further include detecting lipid nanoparticles that do not contain a therapeutic agent. The method may include detecting aggregated lipid nanoparticles.

[0009] According to some embodiments, a method for isolating lipid nanoparticles containing a therapeutic agent may include separating lipid nanoparticles based on size, density, or size and density by CF3 using a carrier solution containing a monosaccharide; detecting lipid nanoparticles containing a therapeutic agent; and isolating the lipid nanoparticles containing a therapeutic agent. In some embodiments, the detecting step may be performed using at least one detector selected from the group consisting of a MALS detector, a photodiode array detector, and an absorbance detector. The monosaccharide may be glucose. In some embodiments, the lipid nanoparticles may contain a therapeutic agent, such as a messenger RNA molecule. The messenger RNA molecule may encode a vaccine. In other embodiments, the therapeutic agent encapsulated in the lipid nanoparticles may be an influenza vaccine, a cancer drug, or an RNAi drug. The method may further include detecting lipid nanoparticles that do not contain a therapeutic agent and / or detecting aggregated lipid nanoparticles. The method may further include discarding lipid nanoparticles detected as not containing a therapeutic agent and / or discarding aggregated lipid nanoparticles. The lipid nanoparticles detected in the methods described herein may not be in contact with an isotonicity agent during the detecting step.The lipid nanoparticles detected in the methods described herein may not be in contact with an isotonicity agent during the detecting step, which suppresses electrostatic interactions between nanoparticles and reduces particle aggregation by reducing the interaction between water and phospholipids.The lipid nanoparticles detected in the methods described herein may not be in contact with a sugar during the detecting step.

[0010] According to some embodiments, a method for administering lipid nanoparticles containing a therapeutic agent may include separating lipid nanoparticles based on size, density, or size and density by CF3 using a carrier solution containing a monosaccharide; detecting lipid nanoparticles containing a therapeutic agent; isolating the lipid nanoparticles containing the therapeutic agent; and administering the isolated lipid nanoparticles containing the therapeutic agent. In some embodiments, the detecting step may be performed using at least one detector selected from the group consisting of a MALS detector, a photodiode array detector, and an absorbance detector. The monosaccharide may be glucose. In some embodiments, the lipid nanoparticles may contain a therapeutic agent, such as a messenger RNA molecule. The messenger RNA molecule may encode a vaccine. In other embodiments, the therapeutic agent encapsulated in the lipid nanoparticles may be an influenza vaccine, a cancer drug, or an RNAi drug. In some embodiments, the carrier solution may be a phosphate buffer. The method may further include detecting lipid nanoparticles that do not contain a therapeutic agent and / or detecting aggregated lipid nanoparticles. The method may further comprise discarding lipid nanoparticles detected as not containing a therapeutic agent and / or discarding aggregated lipid nanoparticles.

[0011] According to some embodiments, a pharmaceutical composition containing isolated lipid nanoparticles containing a therapeutic agent can be prepared according to any one of the methods described above. According to other embodiments, a system for isolating lipid nanoparticles containing a therapeutic agent can include a particle separator that separates lipid nanoparticles based on size, density, or size and density using CF3 with a carrier solution containing a monosaccharide, a particle detector that detects lipid nanoparticles containing a therapeutic agent, and a particle separator that isolates lipid nanoparticles containing a therapeutic agent. In some embodiments, the particle detector can be at least one selected from the group consisting of a MALS detector, a photodiode array detector, and an absorbance detector. [Effects of the Invention]

[0012] According to the present disclosure, lipid nanoparticles can be detected. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows an example overview of a CF3 analysis according to some embodiments of the present disclosure. [Figure 2] 1 shows a schematic diagram of CF3 used in the examples. [Figure 3] Representative CF3 fractograms of four lipid nanoparticle (LNP) samples under optimized conditions are shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention may be more readily understood by reference to the following detailed description of preferred embodiments of the invention. Although different components and methods have been disclosed and described, it is understood, however, that the present invention is not limited to specific preparations, combinations or configurations, conditions, or methods, which may vary and all modifications and changes will be apparent to those skilled in the art. It is also understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0015] In one aspect, the present disclosure provides a method for detecting nanoparticles. The nanoparticles described herein may be lipid nanoparticles, and all methods using lipid nanoparticles can be applied to other types of nanoparticles. In some embodiments, the nanoparticles described herein may include liposomes. In some embodiments, the nanoparticles described herein may exclude liposomes. In some embodiments, the terms detecting, finding, determining, measuring, evaluating, counting, and assessing particles are used interchangeably and include quantitative and / or qualitative determinations. In some embodiments, detection is performed using a sensor. In some embodiments, detection is performed using at least one detector selected from the group consisting of a MALS detector, a photodiode array detector, and an absorbance detector. The term "detection" as used herein may include, but is not limited to, examining physical properties of lipid nanoparticles, such as particle size and particle density. In some embodiments, a detection wavelength of about 100 to about 900 nm, about 190 to about 800 nm, about 200 to about 300 nm, or about 220 to about 240 nm is used. In some embodiments, the detection wavelength is greater than or equal to about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, or 330 nm, and / or less than or equal to about 900, 800, 700, 600, 500, 400, 300, 290, 280, 270, 260, 250, 240 nm.

[0016] As used herein, a "nanoparticle" is a particle having a diameter of less than about 1,000 nm (1 μm). Nanoparticles may comprise various biodegradable or non-biodegradable polymers, lipids, phospholipids, or metals. Lipid nanoparticles according to some embodiments may include, but are not limited to, phospholipids, triacylglycerols, cholesterol, cholesterol esters, and fatty acyl esters. In some embodiments, nanoparticles may comprise a combination of these components. For example, in one preferred embodiment, the lipid core may be made of cholesterol esters and triglycerides (e.g., castor oil), the phospholipid layer may be made of egg yolk phospholipids, and the surfactant coating layer may be made of sodium taurodeoxycholate and poloxamer 188.

[0017] A. Nanoparticle Phospholipids Suitable phospholipids for use in nanoparticles include, but are not limited to, diacylglyceride structures and phosphosphingophospholipids. Diacylglyceride structures include phosphatidic acid (phosphatidic acid ester) (PA); phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (lecithin) (PC), phosphatidylserine (PS), and phosphoinositides. Phosphosphingophospholipids include ceramide phosphorylcholine (sphingomyelin) (SPH), ceramide phosphorylethanolamine (sphingomyelin) (Cer-PE), and ceramide phosphoryllipid. Suitable phospholipids for use in nanoparticle preparations include natural phospholipid derivatives and synthetic phospholipid derivatives. Natural phospholipid derivatives include egg PC, egg PG, soybean PC, hydrogenated soybean PC, and sphingomyelin.Synthetic phospholipid derivatives include phosphatidic acid, phosphatidylcholine, 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), phosphatidylglycerol (PG), 1, 2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG); 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG); 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG); phosphatidylethanolamine (DMPE); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0018] In one embodiment, phospholipids suitable for use in the nanoparticles include 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), phosphatidylglycerol (DMPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), and egg PC. In one embodiment, the phospholipid includes egg PC.

[0019] B. Nanoparticle triglycerides (triacylglycerols) The triglyceride suitable for use in nanoparticle preparation includes (but is not limited to) the triglyceride that is liquid at room temperature.The triglyceride suitable for use in nanoparticles is selected from the group including canola oil, castor oil, chia seed oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, safflower oil, sesame oil and soybean oil. Triglycerides also include mono-, di-, and triacylglycerols, where the fatty acids may be monounsaturated fatty acids (such as palmitoleic acid, oleic acid, elaidic acid, gadoleic acid, eicosenoic acid, and erucic acid), diunsaturated fatty acids (such as linoleic acid, eicosadienoic acid, and docosadienoic acid), and polyunsaturated fatty acids (such as linolenic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, tetracosapentaenoic acid, and docosahexaenoic acid). The di- and triacylglycerols may or may not contain the same fatty acids. Also, classified triglycerides, modified triglycerides, synthetic triglycerides, hydrogenated triglycerides, and mixtures of triglycerides may be used.

[0020] In embodiments, suitable triglycerides for use in the nanoparticles include castor oil, soybean oil, coconut oil, and / or hydrogenated castor oil. In certain embodiments, the triglyceride of the nanoparticles is castor oil, and the therapeutic agent may be dissolved in this component in the nanoparticle core.

[0021] C. Nanoparticle cholesterol and cholesterol esters Cholesterol ester refers to cholesterol esterified with saturated fatty acids, including (but not limited to) myristic acid, palmitic acid, stearic acid, arachidic acid, and lignoceric acid, or with unsaturated fatty acids, including, but not limited to, palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, linolenic acid, arachidonic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, tetracosapentaenoic acid, and docosahexaenoic acid.

[0022] In some embodiments, the cholesterol ester of the nanoparticle is cholesteryl oleate. The cholesterol ester is located in the lipid core, and the cholesterol is located in the phospholipid layer. Cholesterol is typically used in a proportion of 0-4% of the nanoparticle component, i.e., at least 0.1%, at least 0.5%, or at least 1% and at most 3.9%, at most 3.5%, or at most 3%.

[0023] In some embodiments, the lipid nanoparticles have an average lipid concentration of about 60 to about 90%, about 70 to about 90%, about 50 to about 80%, about 40 to about 60%, about 80 to about 95%, about 30 to about 80%, about 60 to about 80%, or about 50 to about 70% by weight, based on the lipid nanoparticles. In some embodiments, the lipid nanoparticles have an average lipid concentration of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90% or more by weight, and / or less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50% by weight, based on the total amount of lipid nanoparticles.

[0024] In some embodiments, the lipid nanoparticles have an average density less than that of the carrier solutions described herein. In some embodiments, the lipid nanoparticles have an average density of about 0.5 to about 1.5 g / ml, about 0.8 to about 1.1 g / ml, 0.9 to about 1.1 g / ml, about 0.5 to about 0.8 g / ml, about 0.5 to about 1.0 g / ml, about 0.3 to about 2.0 g / ml, or about 0.7 to about 1.4 g / ml. In some embodiments, the lipid nanoparticles have an average density of greater than or equal to about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, or 1.2 g / ml and / or less than or equal to about 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0 g / ml. In some embodiments, the particles herein have an average diameter of about 20 to about 130 nm, about 20 to about 50 nm, about 20 to about 150 nm, about 10 nm to about 200 nm, or about 5 nm to 300 nm. In some embodiments, the lipid nanoparticles have an average diameter of about or greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm, and / or less than or equal to about 1000, 900, 800, 700, 600, 500, 400, 300, or 200 nm.

[0025] As used herein, the term "about" is meant to modify values ​​and ranges, such as the length, degree of error, size, processing temperature, processing time, yield, flow rate, and pressure of a nucleotide sequence, and refers to variations in numerical quantities that may arise, for example, from common measuring and handling procedures used in producing a compound, composition, concentrate, or using a preparation, as well as from inadvertent errors in these procedures, differences in the manufacture, source, or purity of starting materials or components used in carrying out such methods, and other considerations. The term "about" also encompasses amounts that vary, for example, due to aging of a composition, preparation, or cell culture at a particular initial concentration or mixture, and amounts that vary due to mixing or processing of a composition or preparation at a particular initial concentration or mixture. The claims appended hereto include equivalents of such quantities, whether modified by the term "about." Furthermore, the term "about" can refer to a range of values ​​similar to a stated reference value. In certain embodiments, the term "about" refers to a range of values ​​that are within 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent of, or less than, the stated reference value.

[0026] In some embodiments, the lipid nanoparticles described herein may be physically separated or "sorted" using CF3 and one of the detection methods described herein. In some embodiments, the CF3 described herein may include high-performance liquid chromatography (HPLC), which may utilize pressure-driven flow of a mobile phase through a column packed with a stationary phase. Based on the physical properties obtained from such detection, it may also be possible to determine whether a particular lipid nanoparticle contains a therapeutic agent and / or whether it is aggregated with other nanoparticles. The lipid nanoparticles described herein may be aggregated. The term "aggregated" lipid nanoparticles, as used in reference to lipid nanoparticles herein, refers to a group of lipid nanoparticles that are stuck together and are no longer an efficient carrier of the therapeutic agent contained therein. "Aggregated" lipid particles according to some embodiments may contain about 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 35, 40, 50, 100, 150, 200, 300, 500, 700, 900, or 1000 lipid nanoparticles. In other embodiments, "aggregated" lipid particles may have diameters greater than or equal to about 30 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1,000 nm, 2,000 nm, 5,000 nm, 10,000 nm, 20,000 nm, or 50,000 nm, and / or less than or equal to about 50 nm, 100 nm, 200 nm, 500 nm, 1,000 nm, 2,000 nm, 5,000 nm, 10,000 nm, 20,000 nm, 50,000 nm, or 100,000 nm. In yet other embodiments, "aggregated" lipid particles may be determined using thresholds for different physical properties.

[0027] As used herein, the term "and / or" is defined as referring to any combination of elements. Also, the singular forms "a," "an," and "the" may further include the plural forms thereof unless clearly stated otherwise.

[0028] In some embodiments, the lipid nanoparticles may contain a therapeutic agent. As used herein, the terms "therapeutic agent," "active agent," or "active ingredient" refer to therapeutically useful amino acids, peptides, proteins, nucleic acids (including, but not limited to, polynucleotides, oligonucleotides, and genes), carbohydrates, and lipids. In some embodiments, the therapeutic agent may include, for example, at least one selected from the group consisting of proteins, enzymes, polysaccharides, polynucleotides, organic compounds, and inorganic compounds. In some embodiments, the therapeutic agent may include neurotrophic factors, growth factors, enzymes, antibodies, neurotransmitters, neuromodulators, antibiotics, antiviral agents, antifungal agents, chemotherapeutic agents, vaccines, and RNAi agents. Therapeutic agents may include drugs, prodrugs, antibiotics, diagnostic agents, imaging agents, and precursors that can be activated upon delivery of the therapeutic agent to target cells or tissues.

[0029] In some embodiments, nanoparticles containing a therapeutic agent may be nanoparticles containing a therapeutic agent in an amount greater than a threshold amount. The threshold amount may be based on the maximum amount that can be encapsulated in the cavity of a given lipid nanoparticle. For example, the threshold amount may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the maximum amount. In some embodiments, the nanoparticles described herein may have a therapeutic agent concentration of about 1% to about 50%, about 10% to about 80%, about 20% to about 50%, about 30% to about 50%, or about 1% to about 10% by weight of the nanoparticle. In some embodiments, the lipid nanoparticles have an average therapeutic agent concentration of about or greater than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90% by weight, and / or about or less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25% by weight, based on the total weight of the lipid nanoparticle.

[0030] Lipid nanoparticles may contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a chemical composition or compound that can be combined with an active ingredient and used to administer the active ingredient to a patient after combination. In some embodiments, "pharmaceutically acceptable carrier" also includes, but is not limited to, one or more of the following: excipients, surfactants, dispersing agents, inert diluents, granulating and disintegrating agents, binders, lubricants, sweeteners, flavoring agents, coloring agents, preservatives, physiologically degradable compositions such as gelatin, aqueous vehicles and solvents, oily vehicles and solvents, suspending agents, dispersing or wetting agents, emulsifiers, demulcents, buffers, salts, thickeners, bulking agents, antioxidants, stabilizers, and pharmaceutically acceptable polymers or hydrophobic materials.

[0031] In some embodiments, an effective amount of a therapeutic agent may be administered to a subject in need thereof. The subject may be a human. As used herein, the term "administration" refers to placing lipid nanoparticles containing a therapeutic agent into a subject by a method or route that can at least partially localize the therapeutic agent at a desired site. Nanoparticles containing a therapeutic agent can be administered in any suitable form and by any appropriate route that results in effective treatment in the subject. As used herein, an "effective amount" refers to an amount sufficient to produce a desired result in an experimental setting. A "therapeutically effective amount" or "therapeutic dose" refers to an amount sufficient to produce a therapeutic response or beneficial clinical outcome in a patient. For methods of some embodiments, a therapeutically effective amount or dose can first be estimated from cell culture assays, and then the dose can be formulated for use in an animal model to achieve a circulating concentration range that includes the IC50 determined in cell culture. This information can then be used to determine a useful dose in the subject. An effective amount of a therapeutic agent can treat a disease in the subject. The disease may be a viral disease and / or COVID-19. The effective treatment may be selected from the group consisting of antipyretics, antivirals, remdesivir, oseltamivir, steroids, plasma containing anti-COVID-19 antibodies from subjects who have recovered from COVID-19, chloroquine, hydroxychloroquine, and a vaccine against COVID-19. In some embodiments, the therapeutic agent may comprise a messenger RNA molecule. In some embodiments, the messenger RNA molecule may encode a vaccine.

[0032] In another aspect, the methods described herein may use CF3. CF3 may be used as a carrier solution. In some embodiments, the carrier solution is alkaline. Furthermore, in some embodiments, the buffer in the carrier solution may be any conventional buffer capable of maintaining a slightly alkaline pH, including, but not limited to, phosphate buffer, Tris buffer, acetate buffer, sulfate buffer, citrate buffer, tartrate buffer, or borate buffer. The carrier solution may have a pH of about 6.5 to about 10, about 6.9 to about 8.0, about 7.0 to about 9.0, about 6.5 to about 9.5, about 7.0 to about 8.5, or about 6.5 to about 8.5. In some embodiments, the pH may be greater than or equal to about 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, or 9.0, and / or less than or equal to about 10.0, 9.8, 9.6, 9.4, 9.2, 9.0, 8.8, 8.6, 8.4, 8.2, 8.0, 7.8, 7.6, 7.4, 7.2, 7.0.

[0033] In some embodiments, the concentration of the buffer in the carrier solution can be from about 1 mmol / L to about 100 mmol / L, from about 1 mmol / L to about 10 mmol / L, from about 10 mmol / L to about 30 mmol / L, from about 5 mmol / L to about 15 mmol / L, from about 20 mmol / L to about 60 mmol / L, or from about 30 mmol / L to about 50 mmol / L. For example, the carrier solution can be a buffer solution having a phosphate concentration greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mmol / L and / or less than about 100, 90, 80, 70, 60, 50, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 mmol / L. Similarly, the solution may contain a buffer having a concentration greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mmol / L and / or less than about 100, 90, 80, 70, 60, 50, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 mmol / L. In some embodiments, the monosaccharide concentration in the carrier solution may be about 1% to about 30%, about 10% to about 20%, about 15% to about 20%, about 10% to about 16%, about 5% to about 20%, or about 13% to about 24% by weight. For example, the carrier solution may contain more than about 1, 2, 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 17.5, 18, 19, or 20% by weight, and / or less than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10% by weight of monosaccharides.Similarly, the solution may contain more than 1, 2, 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 17.5, 18, 19, or 20% by weight and less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10% by weight of another monosaccharide.

[0034] In some embodiments, the carrier solution is flowed through CF3 at a flow rate of about 0.2 mL / min to about 2.5 mL / min, about 0.5 mL / min to about 1.8 mL / min, about 0.7 mL / min to about 1.3 mL / min, or about 0.8 mL / min to about 1.2 mL / min. In some embodiments, the flow rate is greater than or equal to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mL / min and / or less than or equal to 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, or 1.1 mL / min. In some embodiments, the carrier solution is flowed at a temperature of about 50°C to about 70°C. The temperature at which the carrier solution is run may be 20-70° C., 20-50° C., 24-40° C., 24-30° C., or 50-70° C. In some embodiments, the carrier solution is run at a temperature greater than or equal to 20, 24, 30, 40, 50, or 60° C. and / or less than or equal to 100, 90, 80, 70, 60, or 50° C.

[0035] The carrier solution may include a monosaccharide. The monosaccharide may include at least one selected from the group consisting of glucose, fructose, galactose, mannose, xylose, fucose, galactosamine, glucosamine, mannosamine, galacturonic acid, glucuronic acid, iduronic acid, mannuronic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetylmuramic acid, 2-keto-3-deoxy-D-glycero-D-galactonononic acid, N-acetylneuraminic acid, or N-glycolylneuraminic acid. In some embodiments, the monosaccharide may include glucose.

[0036] In some embodiments, the method may include detecting particle size, density, or size and density. Size herein may include particle diameter. Some embodiments of the present disclosure described herein utilize a CF3 to detect and / or sort lipid nanoparticles based on density and / or size. In a CF3, increasing the centrifugal force exerted on the particles may enable the separation of relatively small particles. In some embodiments, the CF3 may be a FFF-C8030 CF3 unit available from Shimadzu Corporation, which has a maximum rotational speed of 12,000 rpm (156,000 m / s 2 A maximum centrifugal force of up to 1000 rpm can be applied. A detailed description of CF3 is provided, for example, in U.S. Patent Application Publication No. 2020 / 0001505, the entire contents of which are incorporated herein by reference. An example overview of CF3 analysis according to some embodiments of the present disclosure is shown in FIG. 1.

[0037] In some embodiments of the present disclosure, CF3 is used to examine the size and / or density of the particles described herein to identify particles containing a therapeutic agent.

[0038] In some embodiments, the methods described herein can sort particles based on size and / or density.The term "sorting" as used herein in relation to particles can include physically separating particles by CF3 based on the physical characteristics of lipid nanoparticles, such as particle size, particle density, and other characteristics, which can serve as a basis for grouping, sorting, or classifying particles.

[0039] In some embodiments, the methods described herein may isolate or purify particles based on size and / or density. As used herein, the terms "isolation" and "purification" may include removing a group of lipid nanoparticles that share a common physical property from a pool of lipid nanoparticles containing another group that has a different physical property. For example, in some embodiments, lipid nanoparticles that are detected to contain a therapeutic agent based on a particular physical property, such as a density value within a predetermined range, may be separated from other lipid nanoparticles that are detected to not contain a therapeutic agent based on a different physical property, such as a density value outside the aforementioned predetermined range. Isolated lipid nanoparticles may be further purified before being administered to a subject. As used herein, the terms "isolation" and "purification" may include removing or discarding lipid nanoparticles that do not contain a therapeutic agent.

[0040] In one aspect, the present disclosure also relates to a pharmaceutical composition prepared according to the methods described herein. The pharmaceutical composition may comprise a therapeutically effective amount of a therapeutic agent described herein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier described herein. In some embodiments, the pharmaceutical composition excludes an isotonic agent, which suppresses electrostatic interactions between nanoparticles and reduces particle aggregation by reducing interactions between water and phospholipids. In some embodiments, the pharmaceutical composition may exclude sugars.

[0041] In one aspect, the present disclosure also relates to a method of administering a particle comprising a therapeutic agent described herein. The method can treat a disease in a subject in need thereof. The subject can be a human. The disease can be a viral disease. The disease can be COVID-19. [Example]

[0042] The following examples further illustrate embodiments of the present disclosure, but do not limit the scope of the disclosure.

[0043] The examples described below discuss a profiling method using CF3 and a multi-angle light scattering detector (CF3-MALS) based on the particle size and density of nanoparticles used in nanomedicines.

[0044] (material) COMIRATY® Intramuscular Injection (Monovalent: Source Strain) (Pfizer), COMIRATY® Intramuscular Injection (Bivalent: Source Strain / Omicron BA.1 and BA.4-5) (Pfizer), and Spikebax® Bivalent (Bivalent: BA.4-5) (Moderna) were obtained from the Ministry of Health, Labor and Welfare of Japan. Reagent-grade disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

[0045] All other reagents were of special or analytical grade. Water was deionized and purified using a Milli-Q® TOC purification system (Millipore, Bedford, MA, USA).

[0046] (Lipid nanoparticle (LNP) sample preparation) Monovalent Comirnaty® intramuscular injection was used as received, thawed, and diluted 1 / 5 with phosphate-buffered saline (Fujifilm Wako, Osaka, Japan). Bivalent Comirnaty® intramuscular injection and Spikebax® were used as received, thawed, and diluted according to the manufacturer's instructions.

[0047] (CF3) A schematic diagram of the CF3 used in this example is shown in Figure 2. CF3 experiments were performed using a Nexera HPLC system (Shimadzu) and an FFF-C8030 (Shimadzu) equipped with a rectangular stainless steel channel (dimensions: 565.5 mm × 20 mm × 0.25 mm (length × width × height) and a turning radius of 99 mm). The HPLC system was equipped with an LC-40D pump, a DGU-405 degassing unit, a SIL-40C autosampler, and an SPD-M40A photodiode array detector (Shimadzu) or an RF-20AXS fluorescence (FL) detector (Shimadzu). MALS detection was performed using a Wyatt DAWN MALS detector (Waters, Milford, MA). Instrument control and data evaluation were performed using LabSolutions LC / GC (Shimadzu) and ASTRA (Wyatt Technology, California, USA) software. UV fractograms of LNPs were recorded in the wavelength range of 190–800 nm using an 8 nm slit with a response time of 1.28 s, a sampling rate of 640 ms, and a wavelength of 230 nm. The cell temperature was set at 40 °C. The angle-dependent scattering data used for MALS detection were evaluated using 14 active angles [light scattering (LS)5–LS18]. The Berry model was used to fit the angle data obtained from the MALS detector.

[0048] The flow rate of the carrier solution or eluent was 1.0 mL / min. The sample suspension was placed in a Shimadzu TORAST-H glass vial (product number: 370-04301-01) and stored at 4 °C in the autosampler until analysis. The carrier solution used for LNP CF3 separation was 10 mmol / L phosphate buffer (pH 8.5) containing 18.4% glucose. The injection volume of LNP samples containing the COMIRATY® Bivalent Origin Strain / Omicron BA.1, COMIRATY® Bivalent Origin Strain / Omicron BA.4-5, and Spikebax® Bivalent was 10 μL. The injection volumes of the COMIRATY® Origin Strain were 2 μL and 10 μL for the undiluted and diluted versions, respectively.

[0049] CF3 separation was carried out under the following conditions: initial rotation speed 12000 rpm (156,000 m / s 2 ), injection time 1 minute, relaxation time (T relax ) 2.5 min, constant field period (T1) 10 min, and decay constant (T a ) - 120 min. Total analysis time (T tot ) is 120 minutes, and rinse time (T rinse ) was 15 minutes.

[0050] Example: CF3 Analysis Conditions for LNPs Containing COVID-19 Vaccines A MALS detector was connected to acquire particle size data and fractograms. To investigate the applicability of CF3 to LNP analysis, Spikebax®, an mRNA LNP, was selected as a representative sample and analyzed using 10 mmol / L phosphate buffer as the carrier solution. As a result, most of the LNP introduced into CF3 eluted in the void peak, making it difficult to retain in CF3. Furthermore, the LNP recovery rate decreased to 72% at neutral pH, but improved at higher pH values. For example, the recovery rates were 81% and 94% at pH 8.5 and pH 12, respectively. However, MALS data suggested that particles might aggregate as the solution pH increased. To improve the recovery rate of LNP from CF3 and enhance LNP retention, a carrier solution was investigated, and 10 mmol / L phosphate buffer (pH 8.5) containing 18.4% glucose was selected as the carrier solution. The actual pH of the carrier solution was 8. The addition of glucose to the phosphate buffer increased the density of the carrier solution, resulting in a significant reduction in the area of ​​the void peak, confirming that LNPs could be retained and properly analyzed by CF3. The reason for this increased retention can be expressed using equation (1).

[0051]

number

[0052] In the above example, the density (ρ l ) increased. Analysis of LNPs with glucose added to the carrier solution showed that the density (ρ l ) than the value of density (ρ ρ ) is considered to be small, so the density (ρ l ) increases, Δ ρ and effective mass (m eff ) increased, indicating that CF3 analysis had the effect of strengthening LNP retention.

[0053] Analytical conditions were optimized using Spikebax® as a representative LNP sample. The recoveries of the samples from the CF3 system (Spikebax®, Comirnaty® original strain, Comirnaty® bivalent original strain / Omicron BA.1, and Comirnaty® bivalent original strain / Omicron BA.4-5) were calculated using peak areas obtained from MALS detection and were 95%, 99%, 92%, and 97%, respectively. Dilution of the Comirnaty® original strain did not affect the elution profile in the fractograms obtained from CF3-MALS or particle size measurements by MALS. Figure 3 shows representative CF3 fractograms of four LNP samples under optimized conditions.

[0054] The particle size distributions for the four samples were in the 20-50 nm and 20-130 nm radius ranges. The CF3 fractogram for sample A in Figure 3 shows that particles with a radius of 120 nm eluted at an elution time of approximately 100 minutes. The CF3 fractogram for sample B showed a low-intensity, broad-shaped peak at an elution time of approximately 100 minutes, indicating the presence of particles similar to the relatively large particles observed in sample A. Furthermore, for samples C and D, no peak was detected at an elution time of approximately 100 minutes in each CF3 fractogram, indicating that the number of particles with a radius of approximately 100 nm was small.

[0055] (Consideration) In the examples described above, CF3 was used to develop an analytical method for profiling injectable nanomedicines by evaluating LNPs of various sizes and with different therapeutic agent contents (densities).

[0056] The density difference between particles and the carrier solution and centrifugal acceleration are two important parameters in CF3 analysis. Unlike liposomes, LNPs may not have a continuous bilayer around the mRNA-lipid matrix, making them soft and resulting in poor physical stability. The challenge of sample recovery from the CF3 system can be overcome by adding glucose to the carrier solution. This can weaken the interaction between LNPs and the channel surface. In addition to liposome products, adding sugars as an isotonic agent to COVID-19 vaccine preparations reduces particle aggregation by suppressing electrostatic interactions between nanoparticles and reducing the interaction between water and phospholipids. Adding glucose to the carrier solution reduces the interaction between particles and the internal surface of the CF3 system, increasing the density of the carrier solution and enhancing LNP retention, enabling proper CF3 analysis. The density of LNPs was lower than that of the carrier solution used in the previous example. The data obtained in the previous example suggest that the combination of CF3 and MALS is a useful methodology for characterizing various nanoparticles based on their size and density.

[0057] In one aspect, the method described herein was developed to analyze nanomedicines containing LNPs based on particle size and density using CF3. Data obtained from CF3 fractograms clearly demonstrate that the proposed methodology can be used to characterize the physicochemical properties of LNPs. In some embodiments, the effect of particle density on in vivo dynamics may be addressed by using CF3 for future nanoparticle characterization. In the future, more effective therapeutic delivery system preparations may be developed using the proposed in vitro particle characterization quantitative analysis. In some embodiments, the method incorporates a wide range of information, including physicochemical and biological characteristics such as particle size distribution, in vitro and in vivo therapeutic release characteristics, in vivo particle diffusion, and stability. This may facilitate the development of innovative and high-quality nanomedicines for improving human health.

[0058] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects. (Section 1) 1. A method for detecting lipid nanoparticles, comprising: A method comprising detecting the size, density, or size and density of lipid nanoparticles by centrifugal field-flow fractionation using a carrier solution comprising a monosaccharide. (Section 2) 10. The method of claim 1, wherein the lipid nanoparticles comprise a therapeutic agent. (Section 3) 3. The method of claim 1 or 2, wherein the lipid nanoparticle comprises a messenger RNA molecule. (Section 4) 4. The method according to any one of items 1 to 3, wherein the lipid nanoparticles contain messenger RNA molecules encoding a vaccine. (Section 5) 5. The method according to any one of items 1 to 4, wherein the lipid nanoparticles contain at least one selected from the group consisting of phospholipids, triacylglycerol, cholesterol, cholesterol esters, and fatty acyl esters. (Section 6) 6. The method according to any one of items 1 to 5, wherein the lipid nanoparticles have an average lipid concentration of 60% to 90% based on the lipid nanoparticles. (Section 7) 7. The method according to any one of items 1 to 6, wherein the lipid nanoparticles have an average density of 0.5 g / mL to 1.5 g / mL. (Section 8) 8. The method according to any one of items 1 to 7, wherein the lipid nanoparticles have an average diameter of 10 nm to 200 nm. (Section 9) 9. The method according to any one of items 1 to 8, wherein the lipid nanoparticles include aggregated lipid nanoparticles. (Section 10) 10. The method according to any one of items 1 to 9, wherein the lipid nanoparticles comprise aggregated lipid nanoparticles having an average diameter of 50 nm to 50,000 nm. (Section 11) 11. The method according to any one of items 1 to 10, wherein the carrier solution contains a phosphate buffer. (Section 12) 12. The method according to any one of items 1 to 11, wherein the carrier solution has a phosphate concentration of 1 mmol / L to 100 mmol / L. (Section 13) 13. The method according to any one of items 1 to 12, wherein the carrier solution is alkaline. (Section 14) 14. The method according to any one of items 1 to 13, wherein the carrier solution has a monosaccharide concentration of 1% by weight to 30% by weight. (Section 15) 15. The method according to any one of items 1 to 14, wherein the carrier solution has a pH of 7.0 to 9.5. (Section 16) 16. The method according to any one of items 1 to 15, wherein the carrier solution has a pH of 7.5 to 9.0. (Section 17) 17. The method according to any one of items 1 to 16, wherein the carrier solution is passed through the centrifugal field-flow fractionation at a flow rate of 0.5 mL / min to 1.8 mL / min. (Section 18) 18. The method according to any one of items 1 to 17, wherein the carrier solution is run at a temperature of 20°C to 70°C. (Section 19) 19. The method according to any one of items 1 to 18, wherein the monosaccharide comprises glucose. (Section 20) 20. The method according to any one of items 1 to 19, wherein the centrifugal field-flow fractionation comprises high performance liquid chromatography. (Section 21) 21. The method according to any one of items 1 to 20, wherein the detecting step is carried out using at least one detector selected from the group consisting of a multi-angle light scattering (MALS) detector, a photodiode array detector, and an absorbance detector. (Section 22) 22. The method according to any one of items 1 to 21, wherein the detecting step is carried out using a MALS detector. (Section 23) 23. The method according to any one of items 1 to 22, wherein the detecting step is carried out at a detection wavelength of 190 to 800 nm. (Section 24) 24. The method according to any one of items 1 to 23, wherein the step of detecting includes a step of detecting the size. (Section 25) 25. The method according to any one of items 1 to 24, wherein the detecting step includes a step of detecting the density. (Section 26) 26. The method according to any one of items 1 to 25, further comprising the step of examining whether the lipid nanoparticles contain a therapeutic agent of interest. (Section 27) 27. The method according to any one of items 1 to 26, further comprising the step of determining the amount of lipid nanoparticles containing the therapeutic agent of interest. (Section 28) 1. A method for sorting or separating lipid nanoparticles, comprising: Detecting lipid nanoparticles according to any one of the methods described in items 1 to 27; and selecting the lipid nanoparticles based on their size, density, or size and density. (Section 29) 29. The method of claim 28, wherein the selecting step results in obtaining a group of lipid nanoparticles that share a common target size and / or target density of lipid nanoparticles. (Section 30) 1. A method for isolating or purifying lipid nanoparticles, comprising: Sorting or separating lipid nanoparticles according to the method of claim 28 or 29; and isolating or purifying the lipid nanoparticles. (Section 31) 31. The method of claim 30, wherein the isolating or purifying step comprises discarding lipid nanoparticles that do not have the therapeutic agent. (Section 32) 32. The method according to claim 30 or 31, wherein the isolating or purifying step results in isolating or purifying a group of lipid nanoparticles that share a common target size and / or target density of lipid nanoparticles. (Section 33) 32. The method of claim 30 or 31, wherein the isolating or purifying step results in the isolation or purification of a population of lipid nanoparticles containing the therapeutic agent. (Section 34) A pharmaceutical composition prepared according to the method of any one of items 30 to 33. (Section 35) 1. A method of administering lipid nanoparticles comprising a therapeutic agent, comprising: Isolating or purifying lipid nanoparticles according to any one of the methods described in items 30 to 33; administering the isolated lipid nanoparticles comprising a therapeutic agent. (Section 36) 1. A method of treating a disease in a subject in need thereof, comprising: 36. Administering lipid nanoparticles according to the method of claim 35, The lipid nanoparticles comprise a pharmaceutically effective amount of a therapeutic agent for treating the disease. (Section 37) 37. The method of clause 36, wherein the disease comprises a viral disease. (Section 38) 37. The method of clause 36, wherein the disease comprises COVID-19. (Section 39) Use of a product or process characterized by one or more elements disclosed in this application.

Claims

1. 1. A method for detecting lipid nanoparticles, comprising: A method comprising detecting the size, density, or size and density of lipid nanoparticles by centrifugal field-flow fractionation using a carrier solution comprising a monosaccharide.

2. The method of claim 1 , wherein the lipid nanoparticle comprises a messenger RNA molecule.

3. 10. The method of claim 1, wherein the lipid nanoparticles comprise messenger RNA molecules encoding a vaccine.

4. 2. The method of claim 1, wherein the lipid nanoparticles comprise at least one selected from the group consisting of phospholipids, triacylglycerols, cholesterol, cholesterol esters, and fatty acyl esters.

5. 10. The method of claim 1, wherein the lipid nanoparticles have an average lipid concentration of 60% to 90% based on the lipid nanoparticles.

6. 10. The method of claim 1, wherein the lipid nanoparticles have an average density of 0.5 g / mL to 1.5 g / mL.

7. The method of claim 1, wherein the lipid nanoparticles have an average diameter of 10 nm to 200 nm.

8. 10. The method of claim 1, wherein the lipid nanoparticles comprise aggregated lipid nanoparticles.

9. 10. The method of claim 1, wherein the lipid nanoparticles comprise aggregated lipid nanoparticles having an average diameter of 50 nm to 50,000 nm.

10. The method of any one of claims 1 to 9, wherein the carrier solution comprises a phosphate buffer.

11. The method of any one of claims 1 to 9, wherein the carrier solution has a phosphate concentration of 1 mmol / L to 100 mmol / L.

12. The method of any one of claims 1 to 9, wherein the carrier solution is alkaline.

13. The method of any one of claims 1 to 9, wherein the carrier solution has a monosaccharide concentration of 1% to 30% by weight.

14. The method of any one of claims 1 to 9, wherein the carrier solution has a pH of 7.0 to 9.

5.

15. The method of any one of claims 1 to 9, wherein the carrier solution has a pH of 7.5 to 9.

0.

16. The method according to any one of claims 1 to 9, wherein the carrier solution is passed through the centrifugal field-flow fractionation at a flow rate of 0.5 mL / min to 1.8 mL / min.

17. The method of any one of claims 1 to 9, wherein the carrier solution is run at a temperature of from 20°C to 70°C.

18. The method of any one of claims 1 to 9, wherein the monosaccharide comprises glucose.

19. The method of any one of claims 1 to 9, wherein the centrifugal field-flow fractionation comprises high performance liquid chromatography.

20. The method according to any one of claims 1 to 9, wherein the detecting step is carried out using at least one selected from the group consisting of a multi-angle light scattering (MALS) detector, a photodiode array detector, and an absorbance detector.

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