Method for detecting particles by using centrifugal field flow grading

Abstract

The present disclosure relates to a method of detecting particles using centrifugal field flow fractionation. The present disclosure provides methods of detecting the size, density, or size and density of lipid nanoparticles. The detection may be performed by centrifugal field flow fractionation using a carrier solution comprising monosaccharides.

Description

[0001] Cross-reference to related applications

[0002] This non-provisional application is based on U.S. Provisional Application No. 63 / 646471, filed with the U.S. Patent and Trademark Office on May 13, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure includes a method for sorting particles by centrifugal field flow fractionation. Background Technology

[0004] Nanomedicines involve the production of nanoparticles containing active pharmaceutical ingredients using nanotechnology, or the formulation of nanoparticles for purposes such as drug delivery. Accurately understanding the relationship between the physicochemical properties of nanoparticles, such as particle size, morphology, and surface properties, and their corresponding biological responses is a significant challenge. Particle size and particle size distribution are considered quality attributes that need to be considered in nanomedicines.

[0005] Currently, electron microscopy and dynamic light scattering (DLS) are used to characterize the size and shape of nanoparticles. In particular, DLS is commonly used in the field of nanomedicines to determine particle size distribution. For example, fractionation methods using various fields such as asymmetric flow, centrifugation, and electro-, thermal, and magnetic field fractionation (FFF) have recently gained considerable attention due to their ability to determine the accurate size distribution of nanoparticles. In FFF techniques, asymmetric flow fractionation (AF4) provides high resolution in the presence of particles of varying sizes in polydisperse mixtures by fractionating nanoparticles based on their diffusion coefficient (i.e., hydrodynamic size). AF4 is increasingly used to determine the size, stability, and drug release capacity of nanomedicines. In contrast, centrifugal FFF (CF3) (also known as sedimentation flow fractionation) can separate particles based on their size and the density difference between the particles and the eluent. Summary of the Invention

[0006] Regarding the physicochemical properties of nanoparticles used as nanomedicines, no evaluation based on particle size and density has been attempted to date. Evaluating the particle size and density of nanoparticles could aid in a complex assessment of their quality and physicochemical properties.

[0007] In one aspect, this document discloses methods for detecting, profiling, sorting, and isolating nanoparticles based on the particle size and density of nanoparticles used in nanomedicines using CF3. The nanoparticles may be lipid nanoparticles. This document further discloses methods for administering lipid nanoparticles isolated and prepared using CF3. Furthermore, pharmaceutical compositions comprising encapsulated therapeutic agents and lipid nanoparticles sorted and / or isolated using CF3 are further disclosed herein.

[0008] Methods for sorting lipid nanoparticles according to some embodiments may include sorting lipid nanoparticles based on size, density, or size and density using CF3 with a carrier solution containing a monosaccharide. The monosaccharide may be glucose. In some embodiments, the lipid nanoparticles may contain therapeutic agents, such as messenger RNA molecules. The messenger RNA molecules may encode vaccines. In other embodiments, the therapeutic agents encapsulated by the lipid nanoparticles may be influenza vaccines, cancer drugs, or RNAi drugs. The carrier solution in some embodiments may be phosphate-buffered saline.

[0009] According to some embodiments, methods for detecting lipid nanoparticles may include sorting lipid nanoparticles based on size, density, or size and density using CF3 with a carrier solution containing a monosaccharide, and detecting lipid nanoparticles containing a therapeutic agent. Detection according to some embodiments may be performed 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. 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 by the lipid nanoparticles may be an influenza vaccine, a cancer drug, or an RNAi drug. The carrier solution in some embodiments may be a phosphate-buffered saline solution. The method may further include detecting lipid nanoparticles that do not contain a therapeutic agent. The method may include detecting aggregated lipid nanoparticles.

[0010] According to some embodiments, methods for separating lipid nanoparticles containing therapeutic agents may include separating lipid nanoparticles based on size, density, or size and density using CF3 with a carrier solution containing monosaccharides, detecting lipid nanoparticles containing therapeutic agents, and separating lipid nanoparticles containing therapeutic agents. Detection according to some embodiments may be performed using at least one 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 by 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 therapeutic agents and / or detecting agglomerated lipid nanoparticles. The method may further include discarding lipid nanoparticles that do not contain therapeutic agents and / or discarding agglomerated lipid nanoparticles. The lipid nanoparticles detected in the methods described herein may not be in contact with an isotonic agent during detection. The lipid nanoparticles detected in the method described herein may not be in contact with an isotonic agent during detection. This isotonic agent inhibits electrostatic interactions between nanoparticles and reduces particle aggregation by decreasing the interaction between water and phospholipids. The lipid nanoparticles detected in the method described herein may not be in contact with sugars during detection.

[0011] 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 using CF3 with a carrier solution containing a monosaccharide, detecting lipid nanoparticles containing a therapeutic agent, separating lipid nanoparticles containing a therapeutic agent, and administering the separated lipid nanoparticles containing a therapeutic agent. Detection according to some embodiments may be performed using at least one 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 by the lipid nanoparticles may be an influenza vaccine, a cancer drug, or an RNAi drug. The carrier solution in some embodiments may be a phosphate-buffered saline solution. The method may further include detecting lipid nanoparticles that do not contain a therapeutic agent and / or detecting agglomerated lipid nanoparticles. The method may further include discarding lipid nanoparticles that do not contain a therapeutic agent and / or discarding agglomerated lipid nanoparticles.

[0012] According to some embodiments, a pharmaceutical composition comprising separated lipid nanoparticles containing a therapeutic agent can be prepared according to any of the foregoing methods. Furthermore, according to other embodiments, a system for separating lipid nanoparticles containing a therapeutic agent may 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 isolator that separates lipid nanoparticles containing a therapeutic agent. The particle detector according to some embodiments may use at least one selected from the group consisting of a MALS detector, a photodiode array detector, and an absorbance detector. Attached Figure Description

[0013] Figure 1 An exemplary overview of CF3 analysis according to some embodiments of this disclosure is depicted.

[0014] Figure 2 A schematic diagram of the CF3 used in the embodiments is depicted.

[0015] Figure 3 Representative CF3 fractograms of four lipid nanoparticle (LNP) samples under optimized conditions were depicted. Figure 3 CF3-MALS data obtained using COVID-19 vaccines. A: Bivalent original strain / Omicron Ba.4-5, B: The original strain, C: Bivalent: Original strain / Omicron BA.1, D: Bivalent: Original strain / Omicron BA.4-5. Detailed Implementation

[0016] The invention can be more readily understood by referring to the following detailed description of preferred embodiments. However, although different components and methods have been disclosed and described, it should be understood that the invention is not limited to specific formulations, components or compositions, conditions, or methods, as these can vary, and any modifications and variations thereof will be apparent to those skilled in the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0017] In one aspect, this 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 not include liposomes. In some embodiments, the detection, discovery, determination, measurement, evaluation, counting, and assessment of 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 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, determining the physical properties of lipid nanoparticles, such as particle size and particle density. In some embodiments, detection wavelengths 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 are used. In some implementations, the detection wavelength is 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 below about 900, 800, 700, 600, 500, 400, 300, 290, 280, 270, 260, 250, 240 nm.

[0018] Here, "nanoparticle" refers to a particle with a diameter of less than about 1,000 nm (1 μm). Nanoparticles can contain 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 contain combinations of these components. For example, in a 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 taurine deoxycholate and poloxamer 188.

[0019] A. Phospholipids in nanoparticles

[0020] Phospholipids suitable for nanoparticle formulations include (but are not limited to) diacylglycerol structures and sphingomyelins. Diacylglycerol structures include phosphatidic acid (phosphatidate) (PA); phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (lecithin) (PC), phosphatidylserine (PS), and inositol phosphatidylphosphate. Sphingomyelins include ceramide phosphocholine (sphingomyelin) (SPH), ceramide phosphoethanolamine (sphingomyelin) (Cer-PE), and ceramide phosphoryl lipid. Phospholipids suitable for nanoparticle formulations 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-dicocanoyl-sn-glycerol-3-phosphocholine (DDPC); 1,2-dilauroyl-sn-glycerol-3-phosphocholine (DLPC); 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC); 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC); 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC); 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC); 1,2-disorcinoyl-sn-glycerol-3-phosphocholine (DEPC); phospholipids Acylglycerols (PG); 1,2-Dimyristoyl-sn-glycerol-3-phosphate glycerol (DMPG); 1,2-Dipalmitoyl-sn-glycerol-3-phosphate glycerol (DPPG); 1,2-distearyl-sn-glycerol-3-phosphate glycerol (DSPG); 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG); phosphatidylethanolamine (DMPE); 1,2-Dimyristoyl-sn-glycerol-3-phosphate ethanolamine; 1,2-dispalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE); 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE); 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

[0021] In embodiments, phospholipids suitable for nanoparticles include 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC); phosphatidylglycerol (DMPG); 1,2-distearatel-sn-glycerol-3-phosphate choline (DSPC); 1,2-distearatel-sn-glycerol-3-phosphate choline (DSPG); and ovarian PC. In one embodiment, the phospholipid comprises ovarian PC.

[0022] B. Triglycerides (triacylglycerols) in nanoparticles

[0023] Triglycerides suitable for nanoparticle formulations include (but are not limited to) triglycerides that are liquid at room temperature. Triglycerides suitable for nanoparticles are selected from the group including rapeseed 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 monoacylglycerols, diacylglycerols, and triacylglycerols, wherein the fatty acids can be monounsaturated fatty acids (palmitoic acid, oleic acid, transoleic acid, gadolinic acid, eicosapentaenoic acid, and erucic acid, etc.), diunsaturated fatty acids (linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid, etc.), and polyunsaturated fatty acids (linolenic acid, di-homo-γ-linolenic acid, eicosatrienoic acid, octadecanoic acid, arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid, etc.). Diacylglycerols and triacylglycerols may or may not contain the same fatty acids. Graded triglycerides, modified triglycerides, synthetic triglycerides, hydrogenated triglycerides, and mixtures of triglycerides can also be used.

[0024] In some embodiments, the triglycerides suitable for the nanoparticles include castor oil, soybean oil, coconut oil, and / or hydrogenated castor oil. In certain embodiments, the triglycerides of the nanoparticles are castor oil, and the therapeutic agent can be dissolved in this component within the nanoparticle core.

[0025] C. Cholesterol and cholesterol esters in nanoparticles

[0026] Cholesterol esters refer to cholesterol esterified with saturated fatty acids (including (but not limited to) myristic acid, palmitic acid, stearic acid, arachidic acid, and lignoceric acid) or unsaturated fatty acids (including but not limited to palmitoleic acid, oleic acid, vaccinic acid, linoleic acid, linolenic acid, arachidonic acid, eicosatrienoic acid, octadecanoic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid).

[0027] In some embodiments, the cholesterol ester of the nanoparticles is cholesterol oleate. The cholesterol ester is located in the lipid core, while cholesterol is located in the phospholipid layer. Cholesterol is typically used in proportions between 0% and 4% of the nanoparticle component, or at least 0.1%, at least 0.5%, or at least 1% and at most 3.9%, or at most 3.5%, or at most 3%.

[0028] In some embodiments, the average lipid concentration of the lipid nanoparticles is about 60 to 90 wt%, about 70 to 90 wt%, about 50 to 80 wt%, about 40 to 60 wt%, about 80 to 95 wt%, about 30 to 80 wt%, about 60 to 80 wt%, or about 50 to 70 wt% based on the total amount of lipid nanoparticles. In some embodiments, the average lipid concentration of the lipid nanoparticles is greater than and / or less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 wt% based on the total amount of lipid nanoparticles.

[0029] In some embodiments, the average density of the lipid nanoparticles is lower than the average density of the carrier solution described herein. In some embodiments, the average density of the lipid nanoparticles is 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 average density of the lipid nanoparticles is above 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 below 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 average diameter of the particles herein is 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 average diameter of the lipid nanoparticles is greater than or equal to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 nm, and / or less than or equal to about 1000, 900, 800, 700, 600, 500, 400, 300, 200 nm.

[0030] As used herein, the term "about" means a change, for example, in the length of a nucleotide sequence, the degree of error, size, amount, concentration, volume, density, diameter, size, processing temperature, processing time, yield, flow rate, and pressure, and its range. It refers to variations in numerical quantities that may occur, for example, through the usual measurement and processing procedures used to prepare compounds, compositions, concentrates, or formulations, through unintentional errors in these processes, through differences in the manufacture, origin, or purity of the starting materials or ingredients used to carry out the method, and similar considerations. The term "about" also covers different amounts resulting from, for example, aging of compositions, formulations, or cell cultures having a particular initial concentration or mixture, and different amounts resulting from mixing or processing compositions or formulations having a particular initial concentration or mixture. Whether or not modified by the term "about," the appended claims include equivalents of these amounts. The term "about" may further refer to a range of values ​​similar to the reference values. In some implementations, the term "about" refers to a range of values ​​falling below 50%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reference value.

[0031] In some embodiments, the lipid nanoparticles described herein can be physically separated or “sorted” using CF3 using one of the detection methods described herein. In some embodiments, the CF3 described herein may include high-performance liquid chromatography (HPLC), which utilizes the pressure of the mobile phase to drive a flow through a column packed with a stationary phase. Based on the physical properties obtained from such detection, it can be further determined whether a particular lipid nanoparticle contains a therapeutic agent and / or whether it aggregates with other nanoparticles. The lipid nanoparticles described herein may be aggregated. The term “aggregated” lipid nanoparticles, used in conjunction with the lipid nanoparticles described herein, means a group of lipid nanoparticles that adhere together such that they are no longer effective carriers of the therapeutic agent contained therein. According to some embodiments, “aggregated” lipid nanoparticles 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, the diameter of the "agglomerated" lipid particles can be 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 other embodiments, thresholds for different physical properties can be used to determine the "agglomerated" lipid particles.

[0032] In this document, the term “and / or” as used herein is defined as referring to any combination of components. Furthermore, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” may further include plural indicators.

[0033] In some embodiments, lipid nanoparticles may comprise therapeutic agents. As used herein, the terms "therapeutic agent," "active agent," or "active ingredient" mean therapeutically useful amino acids, peptides, proteins, nucleic acids (including but not limited to polynucleotides, oligonucleotides, and genes), carbohydrates, and lipids. In some embodiments, therapeutic agents may include at least one selected from the group consisting of proteins, enzymes, polysaccharides, polynucleotides, organic compounds, and inorganic compounds. Therapeutic agents according to some embodiments may include neurotrophic factors, growth factors, enzymes, antibodies, neurotransmitters, neuromodulators, antibiotics, antiviral agents, antifungal agents, chemotherapeutic agents, vaccines, and RNAi drugs. Therapeutic agents may include drugs, prodrugs, antibiotics, diagnostic substances, contrast agents, and precursors that can be activated when the therapeutic agent is delivered to target cells or tissues.

[0034] In some embodiments, the nanoparticles containing the therapeutic agent may be nanoparticles containing the therapeutic agent in an amount exceeding a threshold amount. The threshold amount may be an amount relative to the maximum capacity 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 capacity. In some embodiments, the nanoparticles described herein may contain the therapeutic agent at concentrations based on about 1 wt% to about 50 wt%, about 10 wt% to about 80 wt%, about 20 wt% to about 50 wt%, about 30 wt% to about 50 wt%, or about 1 wt% to about 10 wt%. In some embodiments, based on the total amount of lipid nanoparticles, the average therapeutic concentration of the lipid nanoparticles is greater than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 wt% and / or less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25 wt%.

[0035] Lipid nanoparticles may contain pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition or compound with which an active ingredient can be combined and which, after combination, can be used to administer the active ingredient to a patient. In some embodiments, "pharmaceutically acceptable carriers" also include, but are not limited to, one or more of the following: excipients, surfactants, dispersants, inert diluents, granulators and disintegrants, binders, lubricants, sweeteners, flavorings, colorants, preservatives, physiologically degradable compositions such as gelatin, aqueous solvents and solvents, oily solvents and solvents, suspending agents, dispersants or wetting agents, emulsifiers, demulcents, buffers, salts, thickeners, fillers, antioxidants, stabilizers, and pharmaceutically acceptable polymeric or hydrophobic materials.

[0036] In some implementations, an effective amount of the therapeutic agent can be administered to a subject in need. The subject can be a human. As used herein, the term "administration" means placing a lipid nanoparticle loaded with a therapeutic agent into a subject by a method or route that results in the therapeutic agent being at least partially located at the desired site. Nanoparticles containing the therapeutic agent can be administered in any suitable form and by any suitable route that results in effective treatment in the subject. As used herein, "effective amount" means an amount sufficient to produce the desired result in an experimental setting. "Therapeutic effective amount" or "therapeutic dose" means an amount sufficient to produce a therapeutic response or beneficial clinical outcome in a patient. For methods according to some implementations, the therapeutic effective amount or dose can be initially estimated from a cell culture assay, and then a dose for an animal model can be developed to achieve a range of circulating concentrations including an IC50 as determined in cell culture. Such information can then be used to determine a useful dose in the subject. The effective amount of the therapeutic agent can treat the subject's disease. The disease can be a viral disease and / or COVID-19. Effective treatments may be selected from the group consisting of antipyretics, antiviral drugs, remdesivir, oseltamivir, steroids, plasma containing antibodies against COVID-19 from subjects who have recovered from COVID-19, chloroquine, hydroxychloroquine, and vaccines against COVID-19. In some embodiments, the therapeutic agent may contain a messenger RNA molecule. In some embodiments, the messenger RNA molecule may encode a vaccine.

[0037] On the other hand, the methods described herein may use CF3. CF3 may use 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 pH of the carrier solution may be from about 6.5 to about 10, from about 6.9 to about 8.0, from about 7.0 to about 9.0, from about 6.5 to about 9.5, from about 7.0 to about 8.5, or from about 6.5 to about 8.5. In some implementations, the pH can be above 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, 9.0 and / or below 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.

[0038] In some embodiments, the concentration of the buffer solution in the carrier solution may be about 1 mmol / L to about 100 mmol / L, about 1 mmol / L to about 10 mmol / L, about 10 mmol / L to about 30 mmol / L, about 5 mmol / L to about 15 mmol / L, about 20 mmol / L to about 60 mmol / L, or about 30 mmol / L to about 50 mmol / L. For example, the carrier solution can be a buffer solution with 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 solution with 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. Furthermore, in some embodiments, the monosaccharide concentration in the carrier solution can be from about 1 wt% to about 30 wt%, from about 10 wt% to about 20 wt%, from about 15 wt% to about 20 wt%, from about 10 wt% to about 16 wt%, from about 5 wt% to about 20 wt%, or from about 13 wt% to about 24 wt%. For example, the carrier solution can contain more than about 1, 2, 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 17.5, 18, 19, or 20 wt% 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 wt% 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 wt% 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 wt% of different monosaccharides.

[0039] In some embodiments, the carrier solution is run in 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 above about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mL / min, and / or below 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 run at a temperature of about 50°C to about 70°C. The operating temperature of the carrier solution can be 20 to 70°C, 20 to 50°C, 24 to 40°C, 24 to 30°C, or 50 to 70°C. In some embodiments, the carrier solution is operated at temperatures above about 20, 24, 30, 40, 50, 60°C and / or below 100, 90, 80, 70, 60, 50°C.

[0040] The carrier solution may contain monosaccharides. Monosaccharides may contain at least one selected from the group consisting of glucose, fructose, galactose, mannose, xylose, fucose, galactosamine, glucosamine, mannosamine, galacturonic acid, glucuronic acid, iduronic acid, mannouronic acid, N-acetylgalactosamine, N-acetylglucosamine, N-acetylmannosamine, N-acetylmuramic acid, 2-keto-3-deoxy-D-glycerol-D-galactononanoic acid, N-acetylneuraminic acid, or N-hydroxyacetylneuraminic acid. In some embodiments, the monosaccharide may contain glucose.

[0041] In some embodiments, the method may include detecting the size, density, or size and density of the particles. Size as used herein may include the diameter of the particles. Some embodiments of this disclosure described herein utilize CF3 to detect and / or sort lipid nanoparticles based on density and / or size. In CF3, increasing the centrifugal force applied to the particles can make it possible to separate smaller particles. In some embodiments, the CF3 may be the CF3 unit FFF-C8030 available from Shimadzu Corporation, which can apply up to 12,000 rpm (156,000 m / s). 2 The maximum centrifugal force. A detailed description of CF3 can be found, for example, in U.S. Patent Application Publication 2020 / 0001505, which is incorporated herein by reference in its entirety. Figure 1 An exemplary overview of CF3 analysis according to some embodiments of this disclosure is shown.

[0042] Some embodiments of this disclosure use CF3 to determine the size and / or density of the particles described herein to identify particles loaded with therapeutic agents.

[0043] In some implementations, the methods described herein can sort particles based on size and / or density. The term "sorting," used in conjunction with "particles" herein, can include physically separating particles by means of CF3-based physical characteristics of lipid nanoparticles, such as particle size, particle density, and other characteristics that can be used as a basis for grouping, classifying, or categorizing the particles.

[0044] In some embodiments, the methods described herein may separate or purify particles based on size and / or density. The terms "separation" or "purification" as used herein may include removing a group of lipid nanoparticles having common physical properties from a pool of lipid nanoparticles having properties different from the common physical properties. For example, in some embodiments, lipid nanoparticles detected as containing a therapeutic agent based on a specific physical property, such as a density value falling within a predetermined range, may be separated from other lipid nanoparticles detected as not containing a therapeutic agent based on a different physical property, such as a density value falling outside the aforementioned predetermined range. The separated lipid nanoparticles may be further purified before being administered to a subject. The terms "separation" or "purification" as used herein may include removing or discarding lipid nanoparticles that do not contain a therapeutic agent.

[0045] In one aspect, this disclosure also relates to pharmaceutical compositions prepared according to the methods described herein. The pharmaceutical composition may comprise a therapeutically effective amount of the therapeutic agent described herein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier as described herein. In some embodiments, the pharmaceutical composition does not contain an isotonic agent that inhibits electrostatic interactions between nanoparticles and reduces particle aggregation by reducing interactions between water and phospholipids. In some embodiments, the pharmaceutical composition may not contain sugars.

[0046] In one aspect, this disclosure also relates to a method of administering particles comprising a therapeutic agent as described herein. This method can treat a disease in a subject in need. The subject can be a human. The disease can be a viral disease. The disease can be COVID-19.

[0047] Example

[0048] The embodiments of this disclosure are further explained in the following illustrative examples, but are not limited thereto.

[0049] The examples explained below use CF3 and multi-angle light scattering detector (CF3-MALS) to examine analytical methods for the particle size and density of nanoparticles used in nanomedicine.

[0050] (Material)

[0051] Intramuscular injection (unit price: original strain) (Pfizer) Intramuscular injection (bivalent: original strain / Omicron BA.1 and BA.4-5) (Pfizer) and The divalent (BA.4-5) (Moderna) was obtained from the Japanese Ministry of Health, Labor and Welfare. JIS-grade disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from FUJIFILM Wako Pure Chemical Industries (Osaka, Japan).

[0052] All other reagents are premium or analytical grade. Water is deionized and used... TOC purification system (Millipore, Bedford, MA, USA) purification.

[0053] (Preparation of lipid nanoparticle (LNP) samples)

[0054] The unit price was obtained after thawing and diluting with phosphate-buffered saline (FUJIFILM Wako, Osaka, Japan) at a 1 / 5 ratio. Injectable solution. Use the divalent solution obtained after thawing according to the manufacturer's instructions. Injection and

[0055] (CF3)

[0056] exist Figure 2 A schematic diagram of the CF3 used in this exemplary embodiment is provided. CF3 experiments were performed using an FFF-C8030 (Shimadzu) equipped with a Nexera HPLC system (Shimadzu) and a rectangular stainless steel channel measuring 565.5 mm × 20 mm × 0.25 mm (length × width × height) with a rotation radius of 99 mm. The HPLC system included an LC-40D pump, a DGU-405 degasser, a SIL-40C autosampler, and either an SPD-M40A photodiode array detector (Shimadzu) or an RF-20AXS fluorescence (FL) detector (Shimadzu). A Wyatt DAWN MALS detector (Waters, Milford, MA) was used for MALS detection. Instrument control and data evaluation were performed using LabSolutions LC / GC (Shimadzu) and ASTRA (Wyatt Technology, California, US) software. UV gradation maps of the LNP were recorded in the 190–800 nm wavelength range using an 8 nm slit, a response rate of 1.28 s, a sampling rate of 640 ms, and a wavelength of 230 nm. The cell temperature was set to 40 °C. Angle-correlated scattering data for MALS detection were evaluated using 14 effective angles [Light Scattering (LS) 5–LS 18]. A Berry model was used to fit the angle data obtained from the MALS detector.

[0057] 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 in an autosampler at 4°C until analysis. The carrier solution for CF3 separation of LNP was 10 mmol / L phosphate buffer (pH 8.5) containing 18.4% glucose. Bivalent original strain / Omicron BA.1 Bivalent original strain / Omicron BA.4-5 and The injection volume of the divalent LNP sample was 10 μL. Undiluted and diluted samples were also included. The injection volumes of the original strains were 2 μL and 10 μL, respectively.

[0058] CF3 separation was performed under the following conditions: initial speed of 12,000 rpm (156,000 m / s). 2 The injection time is 1 minute, and the relaxation time (T) is... relax The constant field period (T1) is 10 minutes, and the decay constant (T) is 2.5 minutes. a The analysis time is -120 minutes. Total analysis time (T) tot The time is 120 minutes, and the rinsing time (T) is... rinse (15 minutes)

[0059] (Example: CF3 analysis conditions corresponding to LNPs containing COVID-19 vaccines)

[0060] Connect the MALS detector to obtain particle size data and classification maps. To determine the suitability of CF3 for LNP analysis, select... (A type of mRNA LNP) was used as a representative sample and analyzed using 10 mmol / L phosphate buffer as the carrier solution. The results showed that most of the LNPs introduced into CF3 eluted with the void peak and were difficult to retain for CF3. In addition, the recovery rate of LNP decreased to 72% at neutral pH and improved recovery rate was observed at higher pH values. For example, the recovery rates were 81% and 94% at pH 8.5 and 12, respectively. However, according to MALS data, particle aggregation was suspected as the pH of the solution increased. In order to improve the recovery rate of LNP from CF3 and to improve the retention of LNP, the 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. It was demonstrated that the addition of glucose to the phosphate buffer increased the density of the carrier solution and thus significantly reduced the area of ​​the void peak, making it possible to retain LNP and perform proper fractionation analysis by CF3. The reason for the improved retention can be expressed by equation (1).

[0061]

[0062] The true mass (m) of a particle is determined by its density (ρ). p ) and the density (ρ) of the carrier solution l ) and its effective mass (m eff Related to ) Additionally, Δ ρ This is the density difference between the analyte and the carrier solution. Using Δ... ρ The absolute value is because the density of the analyte may be higher or lower than the density of the carrier solution.

[0063] In the above embodiments, due to the addition of glucose to the phosphate buffer, the density (ρ) l The density (ρ) increases because it is believed that in LNP analysis with the addition of glucose to the carrier solution, the density (ρ) increases. p The value of ) is lower than that of density (ρ) l The value of ) can be clearly understood, therefore, the density (ρ) l The increase of ) leads to Δ ρ and effective mass (m) eff The increase of LNPs improves the retention of LNPs for CF3 analysis.

[0064] use The analytical conditions were optimized using a representative LNP sample. The peak area obtained from MALS detection was used to calculate... Original strain, The bivalent original strain / OmicronBA.1 and The recoveries of the bivalent original strain / Omicron BA.4-5 sample from the CF3 system were 95%, 99%, 92%, and 97%, respectively. (Dilution) The original strain does not affect the elution curves in the grading plots obtained from CF3-MALS and the particle size measurements via MALS. Figure 3 Representative CF3 gradations for four LNP samples under optimized conditions are shown.

[0065] The particle size distribution of the four samples ranged from 20-50 nm (radius) and 20-130 nm (radius). Figure 3 The CF3 fractionation plot of sample A shows that particles with a radius of 120 nm eluted within approximately 100 minutes. In the CF3 fractionation plot of sample B, a broad peak with low intensity was observed at approximately 100 minutes, indicating the presence of particles similar in size to those detected in sample A. Furthermore, in samples C and D, no peaks were detected at approximately 100 minutes in either CF3 fractionation plot, and the number of particles with a radius of approximately 100 nm was small.

[0066] (discuss)

[0067] In the above embodiments, the inventors used CF3 to evaluate LNPs of various sizes and with different therapeutic agent contents (densities) to develop an analytical method for analyzing injectable nanomedicines.

[0068] The density difference between the particles and the carrier solution, as well as centrifugal acceleration, are two important parameters in CF3. Unlike liposomes, LNPs may not possess a continuous bilayer surrounding the mRNA-lipid matrix, making them soft and limiting their physical stability. The sample recovery problem from the CF3 system can be addressed by adding glucose to the carrier solution. This weakens the interaction between the LNPs and the channel surface. In addition to liposome products, sugars have been added as isotonic agents to COVID-19 vaccine formulations to suppress electrostatic interactions between nanoparticles and reduce particle aggregation by reducing the interaction between water and phospholipids. Adding glucose to the carrier solution reduces the interaction between the particles and the inner surface of the CF3 system, improving LNP retention by increasing the density of the carrier solution, which allows for proper CF3 analysis. The density of the LNPs is lower than that of the carrier solution used in the above examples. The data obtained in the above examples demonstrate that the combination of CF3 and MALS is a valuable method for characterizing various nanoparticles based on particle size and density.

[0069] In one aspect, the methods described herein are developed for the analysis of LNP-containing nanomedicines using CF3 based on particle size and density. Data obtained from CF3 grading plots indicate that the proposed method can be used to characterize the physicochemical properties of LNPs. In some embodiments, the effect of particle density on in vivo kinetics can be addressed in the future using CF3 characterization of nanoparticles. Quantitative analysis of the proposed in vitro particle properties enables the development of formulations for future therapeutic delivery systems with superior efficacy. In some embodiments, this method integrates a wide range of information, including physicochemical and biological properties such as particle size distribution, in vitro and in vivo therapeutic release characteristics, in vivo particle diffusion, and stability. This can contribute to the development of innovative and high-quality nanomedicines for improving human health.

[0070] [aspect]

[0071] As those skilled in the art will understand, the above example implementations and embodiments are specific examples of the following aspects.

[0072] (Clause 1)

[0073] A method for detecting lipid nanoparticles, comprising:

[0074] The size, density, or size and density of the lipid nanoparticles were determined by centrifugation field flow fractionation using a carrier solution containing monosaccharides.

[0075] (Clause 2)

[0076] According to the method described in Clause 1, the lipid nanoparticles contain a therapeutic agent.

[0077] (Clause 3)

[0078] According to the method of Clause 1 or 2, the lipid nanoparticles contain messenger RNA molecules.

[0079] (Clause 4)

[0080] The method according to any one of Clauses 1 to 3, wherein the lipid nanoparticles comprise messenger RNA molecules encoding a vaccine.

[0081] (Clause 5)

[0082] The method according to any one of Clauses 1 to 4, wherein the lipid nanoparticles comprise at least one selected from the group consisting of phospholipids, triacylglycerols, cholesterol, cholesterol esters, and fatty acyl esters.

[0083] (Clause 6)

[0084] According to any one of the provisions 1 to 5, the average lipid concentration of the lipid nanoparticles is 60% to 90% based on the lipid nanoparticles.

[0085] (Clause 7)

[0086] According to any one of the provisions 1 to 6, the average density of the lipid nanoparticles is from 0.5 g / mL to 1.5 g / mL.

[0087] (Clause 8)

[0088] The method according to any one of clauses 1 to 7, wherein the lipid nanoparticles have an average diameter of 10 nm to 200 nm.

[0089] (Clause 9)

[0090] The method according to any one of Clauses 1 to 8, wherein the lipid nanoparticles comprise aggregated lipid nanoparticles.

[0091] (Clause 10)

[0092] The method according to any one of Clauses 1 to 9, wherein the lipid nanoparticles comprise aggregated lipid nanoparticles with an average diameter of 50 nm to 50,000 nm.

[0093] (Clause 11)

[0094] The method according to any one of Clauses 1 to 10, wherein the carrier solution comprises phosphate buffer.

[0095] (Clause 12)

[0096] According to any one of the provisions 1 to 11, the phosphate concentration of the carrier solution is from 1 mmol / L to 100 mmol / L.

[0097] (Clause 13)

[0098] The method according to any one of Clauses 1 to 12, wherein the carrier solution is alkaline.

[0099] (Clause 14)

[0100] According to any one of the provisions 1 to 13, the monosaccharide concentration of the carrier solution is from 1 wt% to 30 wt%.

[0101] (Clause 15)

[0102] The method according to any one of Clauses 1 to 14, wherein the pH of the carrier solution is 7.0 to 9.5.

[0103] (Clause 16)

[0104] The method according to any one of Clauses 1 to 15, wherein the pH of the carrier solution is 7.5 to 9.0.

[0105] (Clause 17)

[0106] According to any one of the provisions 1 to 16, the carrier solution is run at a flow rate of 0.5 mL / min to 1.8 mL / min in the centrifugal flow fractionation.

[0107] (Clause 18)

[0108] The method according to any one of Clauses 1 to 17, wherein the carrier solution is operated at a temperature of about 20°C to about 70°C.

[0109] (Clause 19)

[0110] The method according to any one of Clauses 1 to 18, wherein the monosaccharide includes glucose.

[0111] (Clause 20)

[0112] The method according to any one of Clauses 1 to 19, wherein the centrifugal flow fractionation includes high performance liquid chromatography.

[0113] (Clause 21)

[0114] The method according to any one of Clauses 1 to 20, wherein the detection is performed 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.

[0115] (Clause 22)

[0116] The method according to any one of Clauses 1 to 21, wherein the detection is performed using a MALS detector.

[0117] (Clause 23)

[0118] The method according to any one of Clauses 1 to 22, wherein the detection is performed using a detection wavelength of 190 to 800 nm.

[0119] (Clause 24)

[0120] The method according to any one of Clauses 1 to 23, wherein the detection includes the detection of size.

[0121] (Clause 25)

[0122] The method according to any one of Clauses 1 to 24, wherein the detection includes detection density.

[0123] (Clause 26)

[0124] The method according to any one of Clauses 1 to 25 further includes determining whether the lipid nanoparticles contain a therapeutic agent of interest.

[0125] (Clause 27)

[0126] The method according to any one of Clauses 1 to 26 further includes determining the amount of lipid nanoparticles containing the therapeutic agent of interest.

[0127] (Clause 28)

[0128] A method for sorting or separating lipid nanoparticles, the method comprising:

[0129] The lipid nanoparticles were detected according to the method described in any one of Clauses 1 to 27, and

[0130] The lipid nanoparticles are sorted based on size, density, or both size and density.

[0131] (Clause 29)

[0132] According to the method described in Clause 28, the sorting produces a group of lipid nanoparticles with a common target size and / or target density.

[0133] (Clause 30)

[0134] A method for separating or purifying lipid nanoparticles, the method comprising:

[0135] The lipid nanoparticles are sorted or separated according to the method described in Clause 28 or 29; and

[0136] Separate or purify the lipid nanoparticles.

[0137] (Clause 31)

[0138] According to the method described in Clause 30, the separation or purification includes discarding lipid nanoparticles that do not have therapeutic agents.

[0139] (Clause 32)

[0140] The method according to Clause 30 or 31, wherein the separation or purification results in the separation or purification of a group of lipid nanoparticles having a common target size and / or target density.

[0141] (Clause 33)

[0142] The method according to clause 30 or 31, wherein the separation or purification results in the separation or purification of a group of lipid nanoparticles containing therapeutic agents.

[0143] (Clause 34)

[0144] A pharmaceutical composition prepared according to any one of clauses 30 to 33.

[0145] (Clause 35)

[0146] A method for applying lipid nanoparticles containing a therapeutic agent, comprising:

[0147] The lipid nanoparticles are separated or purified according to the method described in any one of clauses 30 to 33; and

[0148] The separated lipid nanoparticles containing the therapeutic agent are applied.

[0149] (Clause 36)

[0150] A method of treating a disease in a subject in need, comprising administering the lipid nanoparticles according to the method of Clause 35, wherein the lipid nanoparticles contain a pharmaceutically effective amount of a therapeutic agent for treating the disease.

[0151] (Clause 37)

[0152] The disease described in the method described in Clause 36 includes viral diseases.

[0153] (Article 38)

[0154] The method described in Clause 36 includes COVID-19.

[0155] (Clause 39)

[0156] The feature is the use of one or more elements of the product or method disclosed in this application.

Claims

1. A method for detecting lipid nanoparticles, comprising: The size, density, or size and density of the lipid nanoparticles were determined by centrifugation field flow fractionation using a carrier solution containing monosaccharides.

2. The method of claim 1, wherein the lipid nanoparticles comprise messenger RNA molecules.

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

4. The method according to 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. The method of claim 1, wherein the average lipid concentration of the lipid nanoparticles is 60% to 90% based on the lipid nanoparticles.

6. The method according to claim 1, wherein the average density of the lipid nanoparticles is from 0.5 g / mL to 1.5 g / mL.

7. The method according to claim 1, wherein the average diameter of the lipid nanoparticles is from 10 nm to 200 nm.

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

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

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

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

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

13. The method according to any one of claims 1 to 9, wherein the monosaccharide concentration of the carrier solution is from 1 wt% to 30 wt%.

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

5.

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

0.

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

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

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

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

20. The method according to any one of claims 1 to 9, wherein the detection is performed 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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