Preparation of samples from lipid nanoparticle encapsulated nucleic acids for precise compositional analysis

Abstract

The present disclosure relates to methods of preparing samples from lipid nanoparticle (LNP) encapsulated nucleic acids using solid phase extraction techniques.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to and the benefit of U.S. Provisional Application No. 63 / 414,131, filed on October 7, 2022. This application also claims priority to and the benefit of U.S. Provisional Application No. 63 / 350,059, filed on June 8, 2022. Each of the foregoing applications is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to a method for preparing a sample from a nucleic acid encapsulated by a lipid nanoparticle (LNP) using a solid phase extraction technique. The method utilizes an adsorbent having an anion exchange function to separate nucleic acids from various lipid components present in such a sample, so that secondary analysis of nucleic acids, lipid components, or both can be performed. Background Art

[0004] It has been found that nucleic acids encapsulated in lipid nanoparticles (LNPs) can be used as therapeutic agents, such as for regulating protein expression or as a new vaccine platform. For example, the current COVID-19 messenger RNA (mRNA) vaccines from Pfizer-BioNTech and Moderna use LNPs to encapsulate mRNA to protect it from destructive enzymes and transfer it to cells. The LNPs used in these COVID-19 vaccines contain 1) ionizable lipids whose positive charges bind to the negatively charged phosphate backbone of mRNA; 2) polyethylene glycol-linked (PEGylated) lipids that help stabilize the nanoparticles; and 3) phospholipids and cholesterol molecules that contribute to the structure of the nanoparticles. Thousands of these lipid components surround the mRNA. In addition, the LNP structure-function and stability properties of various lipid molecules (naturally occurring and synthetic) are being evaluated. These evaluations require optimized stoichiometry. In order to analyze both existing LNP-based nucleic acid drug products and development studies, analytical methods for quantitative lipid components and / or nucleic acids present in such LNP-based nucleic acid products are needed.

[0005] Therefore, this area wishes to provide sample preparation techniques and analytical methods for the nucleic acid encapsulated by LNP, to facilitate separation and quantification of the lipid components present, the nucleic acid present or both. It is also desirable to provide stability, activity / efficacy indication analysis of the nucleic acid drug substance present in the nucleic acid encapsulated by such LNP. Summary of the invention

[0006] The present technology generally relates to a method for preparing a sample from a nucleic acid encapsulated by lipid nanoparticles (LNPs). The method is generally characterized in that a combination of dissolution and solid phase extraction techniques is used to separate a lipid component from a nucleic acid present in the nucleic acid encapsulated by the LNP. In particular, the method of the present technology is characterized in that a sample of the nucleic acid encapsulated by the LNP is dissolved in an organic solvent to provide a solution comprising dissolved lipids and solubilized nucleic acids, and the solution is passed through a weak anion exchange (WAX) solid phase adsorbent. The lipid component is passed through an adsorbent to form a lipid sample, which can be further analyzed to determine composition, stoichiometry, etc. Optionally, nucleic acid can be eluted from an adsorbent to form a nucleic acid sample, which can be further analyzed, subjected to additional processing, or both. Surprisingly, it has been found according to the present disclosure that using certain organic solvents is sufficient to destroy the intermolecular interactions between the lipid component and the nucleic acid, while the WAX ​​adsorbent effectively adsorbs nucleic acids, providing a lipid sample with a high recovery rate, without changing the relative abundance of various lipids present in the nucleic acid encapsulated by the initial LNP, and it is substantially free of nucleic acid and is ready for further analysis. Further surprisingly, by eluting from the WAX ​​adsorbent with an eluent (e.g., aqueous buffer) regulated by pH, nucleic acids can be obtained with a good recovery rate. Advantageously, the nucleic acid sample prepared in this way is substantially free of lipid components, and is a form suitable for further direct analysis. In other embodiments, the nucleic acid retained on the adsorbent can be subjected to nuclease digestion while still on the adsorbent, and the digested nucleic acid components obtained by eluting are analyzed subsequently. The disclosed method is simple and fast to operate, and can accurately analyze all components present in the nucleic acid drug product of, for example, LNP encapsulation. Such analysis can be used to determine or confirm the composition, stoichiometry, purity, stability, etc. of such drug products, and can also find utility in the process of developing new therapeutic agents and vaccines based on lipid nanoparticles.

[0007] Thus, in one aspect, the present technology relates to a method for preparing a sample for composition analysis from a lipid nanoparticle-encapsulated nucleic acid, the method comprising:

[0008] dissolving a sample comprising the lipid particle-encapsulated nucleic acid in an organic solvent to provide a solution comprising dissolved lipids and solubilized nucleic acid; and

[0009] The solubilized nucleic acids were adsorbed onto a weak anion exchange (WAX) solid phase adsorbent.

[0010] In some embodiments, the sample comprising lipid particle-encapsulated nucleic acid is a sample of a formulated LNP-encapsulated nucleic acid drug product.

[0011] In some embodiments, the WAX ​​solid phase adsorbent comprises a surface having disposed thereon an anion exchanger residue having a pKa in the range of about 5 to about 11. In some embodiments, the anion exchanger residue has a pKa in the range of about 8 to about 10.

[0012] In some embodiments, the anion exchanger residue comprises a primary amine, a secondary amine, a tertiary amine, or a combination thereof. In some embodiments, the anion exchanger residue comprises piperazine.

[0013] In some embodiments, the organic solvent comprises C1-C4 alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide, or a combination thereof. In some embodiments, the organic solvent is methanol. In some embodiments, the organic solvent is n-propanol.

[0014] In some embodiments, the organic solvent further comprises an additive selected from the group consisting of: an acid, a base, a detergent, a buffer, a salt, and combinations thereof. In some embodiments, the additive comprises an ionizable cationic base. In some embodiments, the ionizable cationic base comprises ammonium or trialkylammonium ions.

[0015] In some embodiments, the method further comprises removing the dissolved lipids from the WAX ​​solid phase adsorbent, thereby forming a lipid sample that is substantially free of nucleic acids. In some embodiments, removing the dissolved lipids comprises passing the dissolved lipids through the WAX ​​solid phase adsorbent. In some embodiments, removing the dissolved lipids further comprises passing an organic solvent through the WAX ​​solid phase adsorbent and collecting the organic solvent that flows through it to provide one or more eluates; and combining the one or more eluates with the dissolved lipids that have been allowed to pass through the WAX ​​solid phase adsorbent.

[0016] In some embodiments, the method further comprises analyzing the lipid sample by high performance liquid chromatography coupled with a suitable detection method such as tunable dual wavelength UV / visible (TUV) detection, evaporative light scattering, mass spectrometry, charged aerosol, or refractive index.

[0017] In some embodiments, the method further comprises eluting the adsorbed nucleic acid from the WAX ​​solid phase adsorbent, thereby forming a nucleic acid sample that is substantially free of lipids. In some embodiments, eluting comprises flowing an aqueous buffer through the WAX ​​solid phase adsorbent, wherein the aqueous buffer has a pH that is greater than or equal to the pKa of anion exchanger residues of the WAX ​​solid phase adsorbent.

[0018] In some embodiments, the aqueous buffer comprises up to about 50% by volume of a non-aqueous solvent. In some embodiments, the non-aqueous solvent comprises a C1-C4 alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide, or a combination thereof.

[0019] In some embodiments, the aqueous buffer has a pH of about 8 to about 12.5 or about 8.5 to about 12. In some embodiments, the aqueous buffer is an ammonium bicarbonate solution having a pH of about 11.

[0020] In some embodiments, eluting further comprises flowing an organic solvent through the WAX ​​solid phase adsorbent and collecting the organic solvent flowing therethrough to provide one or more eluates; and combining the one or more eluates with the nucleic acid that has been eluted from the WAX ​​solid phase adsorbent with the aqueous buffer.

[0021] In some embodiments, the method further comprises analyzing the nucleic acid sample by high performance liquid chromatography coupled with a suitable detection method such as tunable dual wavelength UV / visible (TUV) detection, evaporative light scattering, mass spectrometry, charged aerosol, or refractive index.

[0022] In some embodiments, the method further comprises digesting at least a portion of the nucleic acids present in the nucleic acid sample with a nuclease.

[0023] In some embodiments, the method further comprises hydrolyzing at least a portion of the nucleic acid present in the nucleic acid to form a mixture of constituent residues.

[0024] In some embodiments, the method includes digesting at least a portion of the solubilized nucleic acids adsorbed on the adsorbent to form an adsorbed mixture of nucleic acid digestion products, wherein the digestion includes contacting the adsorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX ​​solid phase adsorbent to form a nucleic acid digestion sample.

[0025] In some embodiments, eluting comprises flowing an aqueous buffer through the WAX ​​solid phase adsorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of anion exchanger residues of the WAX ​​solid phase adsorbent, and wherein the aqueous buffer comprises up to about 50% by volume of a non-aqueous solvent.

[0026] In some embodiments, the non-aqueous solvent includes a C1-C4 alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide, or a combination thereof.

[0027] In some embodiments, the aqueous buffer has a pH of about 7 to about 12.5 or about 8 to about 12.

[0028] In some embodiments, the method further comprises analyzing the nucleic acid digestion sample by high performance liquid chromatography coupled with a suitable detection method such as tunable dual wavelength UV / visible (TUV) detection, evaporative light scattering, mass spectrometry, charged aerosol, or refractive index. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to provide an understanding of the embodiments of the present technology, reference is made to the accompanying drawings, which are not necessarily drawn to scale. The accompanying drawings are exemplary only and should not be interpreted as limiting the present technology. The disclosure described herein is illustrated in the accompanying drawings by way of example and not limitation.

[0030] Figure 1 is a schematic diagram of a method for preparing a lipid sample according to a non-limiting embodiment of the present disclosure.

[0031] Figure 2 is with Figure 1 Another schematic diagram corresponds to the method shown in , and provides the chemical principles believed to be related to the method.

[0032] Figure 3 is a schematic diagram of an additional method of preparing a sample according to an additional non-limiting embodiment of the present disclosure.

[0033] Figure 4 is a schematic diagram of a method for preparing a nucleic acid digestion sample according to additional non-limiting embodiments of the present disclosure.

[0034] Figure 5A and Figure 5B Depicted is an exemplary chromatographic separation of lipid components in a sample according to non-limiting embodiments of the present disclosure.

[0035] Fig. 6A , Figure 6B and Figure 6C Depicted is an exemplary chromatographic separation of nucleic acids in a sample according to non-limiting embodiments of the present disclosure.

[0036] Fig. 7A An exemplary chromatographic separation of an mRNA sample prepared in pH 7.5 buffer is depicted.

[0037] Figure 7B Depicted from Fig. 7A Exemplary chromatographic separation of a solution phase nuclease digest of an mRNA sample.

[0038] Figure 7C Depicted is an exemplary chromatographic separation of a lipid-encapsulated mRNA sample subjected to solution-phase nucleic acid digestion in a disruption buffer solution.

[0039] Fig. 8A Depicted are exemplary chromatographic separations of nucleic acid digestion samples prepared according to additional non-limiting embodiments of the present disclosure.

[0040] Figure 8B Depicted is an exemplary chromatographic separation of a nucleic acid digestion sample prepared by nucleic acid digestion on an adsorbent of a lipid-encapsulated mRNA sample prepared according to additional non-limiting embodiments of the present disclosure.

[0041] Fig. 9A and Fig. 9B They are Fig. 8A and Figure 8B A magnified view of an exemplary chromatographic separation. DETAILED DESCRIPTION

[0042] Before describing several exemplary embodiments of the present technology, it should be understood that the present technology is not limited to the details of construction or process steps set forth in the following description. The present technology is capable of other embodiments and of being practiced or carried out in various ways.

[0043] definition

[0044] With respect to terms used in this disclosure, the following definitions are provided. Unless the context of the text in which a term appears requires a different meaning, this application will use the following terms as defined below.

[0045] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0046] The term "about" used throughout this specification is used to describe and illustrate small fluctuations. For example, the term "about" can refer to less than or equal to ±5%, such as less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.2%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Whether or not explicitly stated, all numerical values ​​herein are modified by the term "about". Values ​​modified by the term "about" certainly include the specific value. For example, "about 5.0" necessarily includes 5.0.

[0047] As used herein, the term "substantially free" (e.g., with respect to nucleic acid or lipid components) means that the sample contains an amount below the level that will interfere in a subsequent assay or an undetectable amount. The amount may vary depending on the specific component and the assay it is subjected to, but is typically less than about 1%, or less than 0.5%, or less than 0.1%, or less than 0.01%, or less than 0.001%, or even 0% by weight of the component of interest.

[0048] As used herein, the term "lipid" refers to a diverse group of organic compounds, including fats, oils, and certain components of biological membranes, which are characterized by a lack of appreciable interaction with water (i.e., exhibiting hydrophobicity). Lipids encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as other sterol-containing metabolites (e.g., cholesterol). Lipid molecules that may be present in a nucleic acid sample encapsulated by a lipid nanoparticle are further described below.

[0049] The term "nucleic acid" as used herein refers to a linear biopolymer chain composed of a series of nucleotides, which may also be referred to as a "polynucleotide". The term "nucleotide" as used herein refers to a monomer unit composed of the following three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. The chain length (number of nucleotides) of a nucleic acid can vary depending on, for example, the source and intended use. For example, a nucleic acid can contain a relatively short chain (e.g., about 2 to about 20, or about 10 to about 200 nucleotides, commonly referred to as an oligonucleotide), or can contain a much larger chain (e.g., thousands, millions, or more nucleotides).

[0050] The two main classes of nucleic acids are ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). If the 5-carbon sugar is D-ribose, the polymer is RNA, which is formed by a chain of nucleotides connected by phosphodiester bonds between the 3′-hydroxyl group of one ribose unit and the 5′-hydroxyl group of an adjacent ribose unit. The nucleotide nitrogenous bases of RNA are adenine, guanine, cytosine, and uracil. If the 5-carbon sugar is 2-deoxyribose, the polymer is DNA, which is formed by a chain of nucleotides connected by phosphodiester bonds between the 3′-hydroxyl group of one 2-deoxyribose unit and the 5′-hydroxyl group of an adjacent 2-deoxyribose unit. The nucleotide nitrogenous bases of DNA are adenine, guanine, cytosine, and thymine.

[0051] Typically, DNA serves as a repository for the encoded genetic information of a cell or organism. The DNA segment synthesized by the cell encoding a specific protein is a gene. In eukaryotic cells, DNA is organized into a dense protein-DNA complex (chromosome). Typically, RNA in the form of messenger RNA, ribosomal RNA, or transfer RNA replicates genetic information from DNA and plays various roles in forming peptides encoded by DNA.

[0052] References to nucleic acids herein encompass any nucleotide chain, including but not limited to RNA, DNA, oligonucleotides, aptamers, and any analogs or derivatives thereof, such as nucleotides containing modified (i.e., artificial) bases or sugars. In addition, references to RNA and DNA include all forms thereof, such as those that may be present in genomes, chromosomes, histones, or isolated genes, those that occur naturally or as forms artificially introduced into cells or viruses, those that encode genetic information or peptide sequences, and artificial, truncated, and / or fragmentary forms of any of the foregoing forms.

[0053] Embodiments of the present disclosure are now described in detail, it being understood that such embodiments are exemplary only. Such embodiments constitute what the inventors now consider to be the best mode for practicing the present technology. Those skilled in the art will recognize that such embodiments are capable of modification and alteration.

[0054] Synthetic nucleic acids provide new vaccine formats and represent new means of regulating protein expression. Whether based on mRNA or chemically synthesized and purified small oligonucleotides, therapeutic agents containing nucleic acids have proven to have great potential in the field of healthcare. The main breakthrough that helps unlock the potential of nucleic acids as therapeutic agents is the formulation of lipid components into lipid nanoparticles (LNPs) to effectively encapsulate nucleic acids (such as mRNA) and thereby provide a vehicle for the cellular endocytosis of nucleic acids (such as mRNA). Cationic lipids are at the core of this technology. They serve as ion pairs with the negatively charged (phosphate) backbone of nucleic acid molecules and counterions of the negatively charged (phosphate) backbone of nucleic acid molecules, and they are used to stabilize the structure through these intermolecular interactions. Nucleic acids adopt such a structure that can be further constructed with cholesterol and zwitterionic phosphatidylcholine lipids and ultimately constructed with amphiphilic hydrophilic counterparts to produce solid lipid nanoparticles containing nucleic acids. Examples of recent drug products include ONPATTRO TM (Patisiran; Alynylam), a lipid nanoparticle-based short interfering RNA (siRNA) drug for the treatment of polyneuropathy induced by hereditary transthyretin amyloidosis; and the Pfizer-BioNTech and Moderna COVID-19 RNA vaccines.

[0055] Various lipids (naturally occurring and synthetic) are being explored to identify favorable structure-function and stability properties for application in further LNP-based therapeutics. It is important to have analytical methods that accurately indicate the stoichiometry of the lipid and / or nucleic acid components present in a newly designed construct, and to determine the stability and purity of obtainable vaccines and therapeutics.

[0056] At present, the preferred method that the lipid composition of the nucleic acid drug of measuring LNP sealing comprises that the nucleic acid particle of LNP sealing is dissolved in an organic solvent and carries out reversed-phase chromatography and separates, with the detection and quantitative coupling that for example adjustable dual-wavelength ultraviolet / visible light (TUV) detects, evaporative light scattering, charged aerosol or mass spectrometry means carry out.In this type of analysis, destroying lipid-nucleic acid interaction is the most important.Every kind of lipid component is quantitatively dependent on that they are separated into independent chromatographic bands.If the strong intermolecular interaction between the sample components keeps undestroyed, then the quantitative possibility of lipid component (for example, cationic lipid component) is negatively affected, causes insufficient report and inaccurate analysis.

[0057] Similar complications may occur when the analyte of interest is the nucleic acid component of a LNP-encapsulated nucleic acid drug product. For example, if the cationic lipid remains bound to the nucleic acid, the detection of the nucleic acid will be confused by its splitting into one or more additional chromatographic bands corresponding to different ranges of non-covalently bound cationic lipids. In another example, incomplete displacement of lipids from nucleic acids may potentially impair nuclease digestion, which is used to facilitate secondary analysis of nucleic acid drug substances.

[0058] In view of these challenges, it is desirable to provide a sample preparation method that ensures complete disruption of lipid-nucleic acid interactions, providing lipid components and / or nucleic acid samples that are ready for further analysis. Accordingly, the present disclosure provides a method for preparing a sample using an adsorbent having anion exchange functionality. The method effectively and easily separates nucleic acids from various lipid components, enabling a variety of secondary analyses of nucleic acids, lipid components, or both. The method generally includes dissolving the lipid-encapsulated nucleic acids by adding an organic solvent, and separating the lipids from the nucleic acids by using anion exchange solid phase extraction. In Figure 1 A flow chart depicting a general method according to one non-limiting embodiment of the present disclosure is provided in . Each method step and subsequent methods for separation and / or quantification of lipid components, nucleic acids, or both are further described below.

[0059] refer to Figure 1 , a nucleic acid encapsulated by a lipid nanoparticle (LNP) is provided. In some embodiments, the nucleic acid encapsulated by a lipid nanoparticle (LNP) is a pharmaceutical product or a vaccine. In some embodiments, the nucleic acid encapsulated by the LNP is a vaccine for viral infection (such as COVID-19). The nucleic acid encapsulated by the LNP comprises a nucleic acid, which can be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or an analog of any of them (e.g., a synthetic variant, including but not limited to an antisense oligonucleotide (ASO)).

[0060] In some embodiments, the nucleic acid is DNA. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the DNA is double-stranded DNA.

[0061] In some embodiments, nucleic acid is RNA. In some embodiments, RNA is single-stranded RNA. In some embodiments, DNA is double-stranded RNA. In some embodiments, nucleic acid is messenger RNA (mRNA), which is a single-stranded RNA corresponding to the genetic sequence of the gene used as a template during protein synthesis. Recently, mRNA has become an important therapeutic agent for treating viruses and genetic diseases. In some embodiments, nucleic acid is single guide RNA (sgRNA), small interfering RNA (siRNA), small hairpin RN (shRNA), transfer RNA (tRNA) or micro RNA (miRNA).

[0062] In some embodiments, the nucleic acid is a plasmid, which is a small circular nucleic acid molecule found in certain microscopic organisms (e.g., bacteria), and which finds utility in, for example, genetic engineering and gene therapy techniques. The plasmid can be double-stranded DNA, single-stranded DNA, or double-stranded RNA.

[0063] The nucleic acid of LNP sealing comprises multiple lipids.For example, the nucleic acid sample of LNP sealing as disclosed herein generally comprises two, three, four or more different types of lipids, such as one or more phospholipids, one or more PEGylated lipids, one or more ionizable lipids, cholesterol or their combination.Each of these lipids plays a specific function in generation and stable lipid nanoparticles.Especially, cationic lipids have positively charged ammonium groups, which associate with negatively charged phosphate groups in the nucleic acid backbone.

[0064] In some embodiments, the nucleic acid of LNP encapsulation comprises phospholipid.Term " phospholipid " as used herein refers to the amphipathic lipid with negatively charged phosphate group and hydrophobic part.Phospholipid is made up of two fatty acid tails (hydrophobic part) and phosphate group head connected by glycerol, and can be natural or synthetic.The non-limiting example of phospholipid comprises soybean lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, diphosphatidylglycerol, phosphatidylserine, phosphatidylcholine, dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine and hydrogenated or partially hydrogenated phosphatidylcholine. In some embodiments, the phospholipid is phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE) or phosphatidylserine (DOPS). In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-DSPC).

[0065] In some embodiments, the nucleic acid encapsulated by the LNP comprises a lipid modified with polyethylene glycol (PEG), also referred to herein as a "PEGylated lipid", "PEGylated lipid" or "PEGylated lipid"; all three terms are used interchangeably. The term "PEGylated lipid" as used herein refers to a lipid having a polyethylene glycol chain covalently bound thereto. Reference to PEGylated lipids includes PEGylated phospholipids and other lipid classes. In some embodiments, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0066] In some embodiments, the nucleic acid of LNP encapsulation comprises ionizable (for example, cationic) lipid.Term " ionizable lipid " as used herein refers to the lipid with amino group, and this amino group can be protonated to produce ammonium cation under the condition of acidic pH, but keeps charge neutrality at physiological pH.The example of ionizable lipid includes but is not limited to 4-(dimethylamino)-butyric acid, (10Z, 13Z)-1-(9Z, 12Z)-9,12-octadecadiene-1-base-10,13-nineteen-carbondiene-1-base ester (MC3), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octyl nonyl ester (SM-102) and [(4-hydroxybutyl) azanediyl] two (hexane-6,1-diyl) two (2-hexyl decanoate) (ALC-0315). In some embodiments, the ionizable lipid is [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0067] In some embodiments, the LNP-encapsulated nucleic acid comprises a sterol, such as cholesterol.

[0068] The disclosed method comprises dissolving a sample in an organic solvent to provide a solution comprising dissolved lipids and solubilized nucleic acids. Figure 1 , the LNP-encapsulated nucleic acid is treated with an organic solvent to dissolve the LNP-encapsulated nucleic acid, thereby forming a solution comprising solubilized nucleic acid and dissolved lipids. As used herein, the term "organic solvent" refers to any carbon-based molecule that can dissolve or disperse one or more other substances (e.g., one or more components of the LNP-encapsulated nucleic acid). Suitable solvents are typically polar and can be protic or aprotic. Suitable organic solvents include, but are not limited to, alcohols, nitriles, amides, sulfoxides, etc. In some embodiments, organic solvents include alcohols with 1 to 4 carbons (C 1-C 4 alcohols), acetonitrile, dimethylformamide, dimethyl sulfoxide, or a combination thereof. Examples of C 1-C 4 alcohols include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and sec-butanol. In specific embodiments, the organic solvent is n-propanol. The addition of an organic solvent is used to at least partially destroy the interaction between the cationic lipid molecule and the nucleic acid, and to dissolve the remaining lipid components (e.g., cholesterol, phospholipids, PEGylated lipids). In Figure 2 A sketch of the dissolution process is provided in . Figure 2 , the LNP-encapsulated nucleic acid 1 is treated with an organic solvent to provide a solution 2 comprising the nucleic acid, a cationic lipid and other lipids (eg, cholesterol, phospholipids, PEGylated lipids).

[0069] In some embodiments, the method also includes utilizing additives in an organic solvent to promote the destruction of the intermolecular bonds that keep the LNP particles together. Suitable additives include, but are not limited to, detergents, buffers, salts, acids, alkalis (including ionizable cationic bases) and combinations thereof. In some embodiments, additives include or are salts of ammonium hydroxide, acetic acid or formic acid, triethylamine, Tris, HEPES, phosphates, Bis-Tris, tetramethylammonium, hexylamine, diisopropylethylamine, trialkylamines, ammonium bicarbonate, ammonium acetate, ammonium formate or combinations thereof. In some embodiments, additives are present in an organic solvent in the form of an aqueous solution. In some embodiments, the aqueous solution, an organic solution further comprising an additive or both have a pH value in the range of about 6 to about 12, such as from about 6, about 7, about 8 or about 9 to about 10, about 11 or about 12. In some embodiments, the aqueous solution, an organic solvent further comprising an aqueous solution or both have a pH in the range of about 6 to about 9. In some embodiments, the organic solvent also comprises triethylammonium acetate. In some embodiments, the organic solvent is a mixture of n-propanol and 0.1 M triethylammonium acetate (pH 10 buffer).

[0070] The method involves adsorption of the solubilized nucleic acids onto a weak anion exchange (WAX) solid phase extraction (SPE) sorbent. Figure 1 , the resulting solution is loaded onto a WAX SPE adsorbent bed. The lipids are allowed to flow through (elute) and form a lipid sample, while the nucleic acids are adsorbed on the adsorbent. Without wishing to be bound by theory, it is believed that the adsorption of nucleic acids on the WAX ​​adsorbent anion exchanger ligands serves to displace and purify away cationic lipids. Further references Figure 2 It is believed that the cationic groups on the surface of the WAX ​​adsorbent associate with the negatively charged phosphate groups in the nucleic acids, resulting in the nucleic acids being adsorbed (retained) 3 on the adsorbent. In contrast, cationic and uncharged lipids are not retained by the adsorbent and are either passed through or further washed through (eluted) with a solvent (e.g., an organic solvent as described herein, and which may be the same or different from the organic solvent used for solubilization) to provide a lipid sample 4.

[0071] In some embodiments, the method also includes analyzing lipid samples, such as by suitable liquid chromatography.For example, the lipid sample comprising lipid components (that is, lipids not retained and / or eluted) can be analyzed to measure their relative abundance, research impurities that may be present in the sample or both.Suitable liquid chromatography includes high performance liquid chromatography and ultra-high performance liquid chromatography coupled with various detection methods, and the detection method includes but is not limited to adjustable dual-wavelength ultraviolet / visible light (TUV) detection, evaporative light scattering, mass spectrometry, charged aerosol or refractive index.

[0072] The WAX ​​adsorbent used can vary. Generally, suitable adsorbents include those having weakly basic to strongly basic anion exchanger residues disposed on the adsorbent surface. Examples of suitable anion exchanger residues include, but are not limited to, primary amines, secondary amines, and tertiary amines and combinations thereof. In some embodiments, the anion exchanger residues include tertiary amino groups. In some embodiments, the anion exchanger residues have a pKa in the range of about 5 to about 11, such as from about 5, about 6, about 7, or about 8 to about 9, about 10, or about 11. In some embodiments, the anion exchanger residues have a pKa in the range of about 8 to about 10. Without wishing to be bound by theory, it is believed that when matching the basicity and hydrophobicity of nucleic acids, selecting an adsorbent containing anion exchanger residues with a specific pKa range and / or hydrophobicity degree can provide enhanced nucleic acid recovery. In some embodiments, the WAX ​​adsorbent comprises polyethyleneimine, diethylaminoethyl, diethylaminopropyl, or dimethylaminopropyl residues. In some embodiments, the WAX ​​adsorbent comprises piperazine residues.

[0073] WAX adsorbents include adsorbent particles. The composition of the adsorbent particles can vary. For example, suitable adsorbent materials include silica, organosilicon, and polymer particles that contain or are modified (e.g., by bonding or grafting) to contain anionic residues as described herein.

[0074] The particle size of the adsorbent may vary. In some embodiments, the adsorbent has a particle size in the range of about 10 μm to about 100 μm in diameter, such as about 20 μm to 70 μm in diameter.

[0075] Solid phase adsorbents are typically porous materials. The pore size of the adsorbent can vary. In some embodiments, the adsorbent has a pore size of about to about In some embodiments, the adsorbent has an average pore size in the range of about to about The average pore size in the range of pore size is 0.1-1.0. Without wishing to be bound by theory, it is believed that the selection of pore size can affect adsorption and elution behavior, depending on the specific nucleic acid present. For example, in some embodiments, a multimodal retention mechanism can occur between the adsorbent and the nucleic acid. Specifically, a larger pore size can allow some nucleic acids to be partially or entirely adsorbed on the particle inner surface and experience a significant amount of restricted diffusion when eluting.

[0076] In some embodiments, the WAX ​​adsorbent comprises porous polymer particles modified with tertiary amino groups having an average pore size of about 80 μm in diameter. A suitable example of such a WAX adsorbent is Oasis TMThe adsorbent is available from Waters, Inc. (Milford, MA) in the form of cartridges or multiwell plates. In other embodiments, the adsorbent is Sep-Pak TM Aminopropyl (Waters, Milford, MA), Poros 50 D (dimethylaminopropyl, ThermoFisher Scientific, Waltham, MA), Poros 50 PI (polyethyleneimine, ThermoFisher Scientific, Waltham, MA), UniDEAE-30S (Suzhou Nanovita Technology Co., Ltd., China), UniBPC 55-WAX (Suzhou Nanovita Technology Co., Ltd., China), UniDEAE-50XS (Suzhou Nanovita Technology Co., Ltd., China), silica bonded to polyethyleneimine (WAX, Separation Methods Technologies, Newark, DE), silica bonded to diethylaminoethyl ligands (DEAE, Separation Methods Technologies, Newark, DE), or Clarity OTX (Phenomenex, Torrance, CA).

[0077] In some embodiments, the lipid components in the lipid sample are obtained at a recovery rate of greater than about 80% by area based on the total area of ​​the lipid components present in the nucleic acid encapsulated by LNP (i.e., in the sample before the disclosed method is performed). In some embodiments, the relative abundance of the lipids present is unchanged in the lipid sample relative to the abundance of the lipid components present in the nucleic acid encapsulated by LNP (i.e., in the sample before the disclosed method is performed).

[0078] In some embodiments, the method further comprises eluting the adsorbed nucleic acid from the WAX ​​solid phase adsorbent, thereby forming a nucleic acid sample that is substantially free of lipids. Figure 2 Typically, the adsorbed nucleic acids are eluted using an eluent having a pH equal to or higher than the pKa of the cationic residues on the WAX ​​adsorbent to provide a nucleic acid sample 5 .

[0079] The eluent for eluting the adsorbed nucleic acid can vary, but is generally a buffered aqueous solution with a high pH (e.g., greater than about 7). In some embodiments, the eluent is an aqueous buffer having a pH in the range of about 8 to about 12.5 (such as from about 8 or about 9 to about 10, about 11 or about 12). In some embodiments, the eluent is an aqueous buffer having a pH in the range of about 8.5 to about 12 (such as about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12). Suitable buffers include but are not limited to aqueous ammonium bicarbonate, ammonium acetate, ammonium formate, triethylamine, tetramethylammonium, hexylamine, diisopropylethylamine and ammonium hydroxide. In a particular embodiment, the eluent is a 100 mM ammonium bicarbonate buffer having a pH of about 11. In some embodiments, the eluent also comprises a non-aqueous solvent. The non-aqueous solvent can exist with an aqueous buffer in various ratios, and such ratios can be determined experimentally by those skilled in the art. In some embodiments, the eluent comprises up to about 50% by volume of a non-aqueous solvent. In some embodiments, the non-aqueous solvent is an organic solvent as described herein. Thus, in some embodiments, the eluent further comprises an organic solvent selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol, n-butanol, and acetonitrile.

[0080] After the initial elution, one or more washings of the adsorbent may be performed to improve the recovery of the nucleic acid. Therefore, in some embodiments, the method further comprises flowing an organic solvent as described herein through the WAX ​​solid phase adsorbent and collecting the organic solvent flowing through it to provide one or more eluates. In some embodiments, the one or more eluates are combined with each other and / or with the nucleic acid fraction of the initial elution. When utilized, such washings may be optimized based on the properties of the adsorbent, nucleic acid, or sample as a whole. For example, washing conditions may be optimized to accommodate sample hydrophobicity, matrix effects, lipid composition, or the presence of polymers and excipients in the sample. Optimization of washing may include adding an acid or base, using an alternative protic or aprotic organic solvent (e.g., ethanol, propanol, DMSO), or applying a solvent mixture such as methanol and acetonitrile in various ratios.

[0081] In some embodiments, the total area of ​​nucleic acid present in the nucleic acid encapsulated by LNP (i.e., in the sample before performing the disclosed method), the disclosed method achieves a nucleic acid recovery rate of 50% or greater by area. In some embodiments, the total area of ​​nucleic acid present in the nucleic acid encapsulated by LNP, the disclosed method achieves a nucleic acid recovery rate of 80% or greater by area.

[0082] In some embodiments, the method further comprises analyzing the nucleic acid sample obtained by elution from the WAX ​​adsorbent. In some embodiments, the method further comprises processing the nucleic acid sample, and optionally, performing various analyses on the processed sample. Figure 3 Schematic diagrams of some of these potential analyses and processes, according to non-limiting embodiments, are provided in .

[0083] In some embodiments, the method further comprises analyzing the nucleic acid sample, such as by a suitable liquid chromatography method. Suitable liquid chromatography methods include high performance liquid chromatography and ultra-high performance liquid chromatography coupled with various detection methods, including but not limited to adjustable dual wavelength ultraviolet / visible light (TUV) detection, evaporative light scattering, mass spectrometry, charged aerosols or refractive index.

[0084] In some embodiments, the method further comprises digesting at least a portion of the nucleic acids present in the nucleic acid sample with a nuclease. The digestion products include, but are not limited to, small (e.g., less than about 20 nucleotides) oligonucleotides, such as oligoribonucleotides. Examples of suitable nucleases include, but are not limited to, RNase T1 and Maz F. In some embodiments, the method further comprises analyzing the digested nucleic acid sample, such as by a suitable liquid chromatography method. Suitable liquid chromatography methods include high performance liquid chromatography and ultra-high performance liquid chromatography in combination with various detection methods, including, but not limited to, UV, evaporative light scattering, mass spectrometry, charged aerosols, and refractive index.

[0085] In some embodiments, the method further comprises performing sequence mapping on the digested nucleic acid mixture.

[0086] In some embodiments, the method further comprises hydrolyzing at least a portion of the nucleic acids present in the nucleic acid sample to form a mixture of component residues. In some embodiments, the method further comprises analyzing the component residues obtained by hydrolysis (e.g., by performing liquid chromatography to separate and / or quantify the bases and / or sugars present in the mixture).

[0087] In other embodiments, the method further comprises digesting at least a portion of the solubilized nucleic acids adsorbed on the adsorbent, thereby forming an adsorbed mixture of nucleic acid digestion products, wherein the digestion comprises contacting the adsorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX ​​solid phase adsorbent, thereby forming a nucleic acid digestion sample. Figure 4 A flow chart depicting a method according to a non-limiting embodiment of the present disclosure is provided in . Each additional method step is further described below.

[0088] Continue to refer Figure 4, in some embodiments, the adsorbed nucleic acid is subjected to digestion with a nuclease while remaining adsorbed on an adsorbent. Surprisingly, it has been found according to the present disclosure that contacting the nucleic acid adsorbed by the adsorbent with a nuclease results in the digestion of at least a portion of the bound nucleic acid, and the resulting digestion spectrum is comparable to the digestion spectrum obtained during the digestion and analysis of the mRNA itself. This discovery makes it possible to quickly process the destroyed LNP mRNA without the need for further precipitation techniques. As described above, in order to destroy LNP, an alcohol solution is generally required, and when carried out in the absence of the described WAX separation method, there is a problem of enzymatic digestion. In particular, the components of lipid nanoparticles are also dissolved in the solution, complicating the analysis. In addition, as demonstrated in Example 2 herein, when carried out directly in the destruction solution, the nuclease digestion of the mRNA sample failed.

[0089] In a non-limiting embodiment of the disclosed method, and with reference to Figure 1 , Figure 2 and Figure 4 , disrupting the LNP-nucleic acid preparation, applying the mixture to a WAX adsorbent, and removing the lipid component, each as described above. However, in this particular embodiment, the adsorbed nucleic acid is digested while remaining adsorbed on the WAX ​​adsorbent. Figure 4 After digestion, the mixture of nucleic acid digestion products thus obtained is eluted from the adsorbent using an eluent, thereby forming a nucleic acid digestion sample.

[0090] In some embodiments, eluting the mixture of nucleic acid digestion products includes passing an aqueous buffer through the WAX ​​solid phase adsorbent. The aqueous buffer used to elute the adsorbed nucleic acids can vary, but is typically an aqueous buffer having a pH greater than or equal to the pKa of the anion exchanger residue of the WAX ​​solid phase adsorbent. In some embodiments, the aqueous buffer has a pH in the range of about 8 to about 12.5 (such as from about 8 or about 9 to about 10, about 11 or about 12). In some embodiments, the aqueous buffer has a pH in the range of about 8.5 to about 12 (such as about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5 or about 12). Suitable buffers include, but are not limited to, aqueous ammonium bicarbonate, ammonium acetate, ammonium formate, triethylamine, tetramethylammonium, hexylamine, diisopropylethylamine and ammonium hydroxide.

[0091] In some embodiments, the aqueous buffer further comprises a non-aqueous solvent. The non-aqueous solvent can exist with the aqueous buffer in various ratios, and such ratios can be determined experimentally by those skilled in the art. In some embodiments, the eluent comprises up to about 50% by volume of the non-aqueous solvent. In some embodiments, the non-aqueous solvent is an organic solvent as described herein. In some embodiments, the aqueous buffer further comprises C1-C4 alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide or a combination thereof.

[0092] In some embodiments, the method further comprises analyzing the nucleic acid digestion sample to provide separated and / or quantitative nucleic acid digestion products. Suitable methods for analyzing the sample include, but are not limited to, high performance liquid chromatography techniques such as UPLC, reversed phase HPLC, ion pairing HPLC, etc., in combination with various detection methods, including but not limited to adjustable dual wavelength ultraviolet / visible (TUV) detection, evaporative light scattering, mass spectrometry, charged aerosols, or refractive index.

[0093] Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the materials and methods and is not intended to limit the scope. Language in the specification should not be construed to indicate that any unclaimed element is essential to the practice of the disclosed materials and methods.

[0094] It will be apparent to those skilled in the relevant art that suitable modifications and changes may be made to the compositions, methods and applications described herein without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of the claimed embodiments. The various embodiments, aspects and options disclosed herein can all be combined in all variations. The scope of the compositions, formulations, methods and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples and preferred embodiments herein.

[0095] Although the technology herein has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the technology. It will be apparent to those skilled in the art that various modifications and variations may be made to the methods and apparatus of the technology without departing from the spirit and scope of the technology. Therefore, the technology is intended to include modifications and variations within the scope of the appended claims and their equivalents.

[0096] References throughout this specification to "one embodiment," "certain embodiments," "one or more embodiments," or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present technology. Thus, the appearance of phrases such as "in one or more embodiments," "in certain embodiments," "in one embodiment," or "in an embodiment" in various places in this specification are not necessarily referring to the same embodiment of the present technology. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. Any ranges cited herein are included.

[0097] Various aspects of the present technology are more fully described with reference to the following examples. Before describing several exemplary embodiments of the present technology, it should be understood that the present technology is not limited to the details of the construction or process steps set forth in the following description. The present technology can have other embodiments and can be practiced or carried out in various ways. The following examples are set forth to illustrate certain aspects of the present technology and should not be construed as limiting thereof.

[0098] Example

[0099] The invention is further illustrated by the following non-limiting examples describing chromatography apparatus and methods.

[0100] Material

[0101] Unless otherwise noted, all reagents were used as received. One skilled in the art will recognize that equivalents to the following supplies and suppliers exist, and therefore, any supplier listed below should not be construed as limiting.

[0102] Example 1. Sample preparation and fractionation of lipids and mRNA

[0103] Samples representing mRNA formulated with lipid nanoparticles (LNPs) were produced from a mixture of lipid components and nucleic acids. Nucleic acids were erythropoietin (EPO) transcripts based on 5-methoxyuridine-modified mRNA (L-7209; available from TriLink Biotechnologies, San Diego, CA). The mRNA stock solution was prepared as a 100 μg / mL solution in 1 mM sodium citrate buffer (pH 6.4). The lipid component consisted of cholesterol (41.9%), an ionizable lipid component (ALC-0315; 45.4%), a pegylated lipid component (ALC-0159; 3.5%), and a phospholipid component (1,2-DSPC; 9.2%). The components were obtained from Cayman Chemical, Ann Arbor, MI (Lipid Nanoparticle Exploration Kit; Product No. 35426). A lipid stock solution was generated from this mixture and contained 1.65 mg / mL cholesterol, 3.54 mg / mL ionizable lipid component, 0.78 mg / mL PEGylated lipid component, and 0.74 mg / mL phospholipids. To form a sample mixture for fractionation, a portion of the lipid stock mixture (10 uL) and a portion of the mRNA stock solution (10 uL) were added to 80 uL of a solvent mixture (36% by volume aqueous triethylammonium acetate (100 mM; pH 10) and 64% by volume n-propanol).

[0104] Oasis TM The wells of a weak anion exchange (WAX) μElution plate (available from Waters, Inc., Milford, MA) were conditioned for use by washing with 2% formic acid in methanol (2×200 μL) followed by washing with 2% formic acid in water (2×200 μL). The sample mixture (100 δ μL) was applied to the wells and the lipids were collected in the pass-through fraction.

[0105] The lipid mixture was then analyzed by reverse phase high performance liquid chromatography (HPLC) using evaporative light scattering detection. The injection volume was 5 μL. A commercially available HPLC system (ACQUITY TM UPLC TMH-Class Plus system, including ACQUITY photodiode array detector and ACQUITY evaporative light scattering detector; all available from Waters Corporation, Milford, MA) for separation. The column was Premier CSH phenyl-hexyl (1.7 μm 2.1×50 mm). Elution was performed as follows: a linear gradient of 100% mobile phase A to 100% mobile phase B was used in 6 minutes at a temperature of 50° C. and a flow rate of 0.4 mL / min, followed by a 2-minute hold at 100% mobile phase B. Mobile phase A was acetonitrile / isopropanol / 0.1% aqueous formic acid in water; 50 / 10 / 40 by volume). Mobile phase B was acetonitrile / isopropanol / 0.1% formic acid in water; 80 / 10 / 10 by volume).

[0106] The LC analysis results of lipid samples are provided in Figure 5A and Figure 5B middle. Figure 5A is a reference chromatogram of the lipid mixture as it exists in the lipid stock mixture (ie, before being subjected to the solid phase extraction procedure). Figure 5B is a chromatogram of a lipid sample obtained by fractionation. Figure 5A and Figure 5B Both, the peak intensities for each lipid component present are provided as follows: Peak #1 is the ionizable lipid; Peak #2 is cholesterol; Peak #3 is the PEGylated lipid; and Peak #4 is the phospholipid. Figure 5A and Figure 5B , it can be observed that each lipid originally present in the sample was recovered in the pass-through fractions with a recovery rate of 84%. In addition, the extraction procedure did not change the relative abundance of each lipid.

[0107] The WAX ​​elution plate unit containing the adsorbed mRNA was then treated to provide the mRNA component. Specifically, the elution plate was washed with methanol (2×50 μL), 2% formic acid in water (2×50 μL), and methanol (2×50 μL) to ensure complete removal of the lipid component. The mRNA was then eluted with aqueous ammonium bicarbonate (100 mM; pH 11) as an eluent (2×50 μL), followed by methanol (2×50 μL).

[0108] The eluents were analyzed by ion-pairing reversed-phase high performance liquid chromatography with UV detection at 260 nm. The injection volume was 10 μL. A commercially available HPLC system (ACQUITY TM UPLC TMH-Class Bio system, equipped with ACQUITY photodiode array detector; each available from Waters Corporation, Milford, MA) for separation. The column was Premier BEH C18 (1.7 μm, 2.1×50 mm). Elution was performed as follows: a linear gradient of 90% mobile phase A to 55% mobile phase B in 3 minutes at a temperature of 50° C. and a flow rate of 0.4 mL / min, followed by a 1 minute hold at 55% mobile phase B. Mobile phase A was 5 / 90 / 5 acetonitrile / water / 1M triethylammonium acetate (pH 6.9) by volume. Mobile phase B was 60 / 35 / 5 acetonitrile / water / 1M triethylammonium acetate (pH 6.9) by volume.

[0109] The results of LC analysis of mRNA samples are provided in FIG. 6A to FIG. 6C middle. Fig. 6A is a reference chromatogram of mRNA as it exists in the mRNA stock solution (ie, before being subjected to the solid phase extraction procedure). Figure 6C is a chromatogram of an mRNA sample eluted from a WAX elution plate unit. Figure 6C , 53% of the mRNA was recovered in the elution fractions (45.3% in the aqueous buffer eluent, and 7.4% in the methanol eluent; data not shown). Figure 6B is a chromatogram of the lipid pass sample showing complete adsorption of the mRNA to the WAX ​​adsorbent as evidenced by the clean baseline.

[0110] Example 2. Attempted nuclease digestion of mRNA formulated with lipid nanoparticles (LNPs) in a disrupting solution

[0111] The messenger RNA (mRNA; firefly luciferase transcript) formulated in lipid nanoparticles (LNP-mRNA; 100 micrograms) was contacted with a disrupting solution (an amine-containing alcohol solution, specifically, 35.7% of 0.1 mM triethylammonium acetate pH 10 buffer in n-propanol), followed by the addition of an endonuclease (RNase T1; excess, about 30 kU for 10 μg mRNA). The resulting mixture was analyzed by ion-pairing reversed phase liquid chromatography and mass spectrometry (IP-RPLC-MS). The separation conditions were as follows:

[0112] LC :

[0113] System: ACQUITY TM UPLC TM Premier system (available from Waters Corporation, Milford, MA)

[0114] Column: ACQUITY TMPremier Peptide BEHTM C18, 2.1×150mm, 1.7μm

[0115] Column temperature: 70.0℃

[0116] Solvent A: 1% 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 0.1% N,N-diisopropylethylamine (DIPEA) in water

[0117] Solvent B: 0.075% HFIP / 0.0375% DIPEA in 65% methanol 35% water

[0118] MS :

[0119] Instrument: Vion IMS QTof

[0120] Instrument driver version: 4.0.0

[0121] Negative mode ESI

[0122] ·MS E , mass range 50m / z-4000m / z, scan time: 0.5 seconds

[0123] Low collision energy: 6V, high collision energy ramp: 10V-30V

[0124] Desolvation temperature: 600°C, source temperature: 150°C

[0125] Lock calibration: Single reference mass at 554.2615 m / z

[0126] Separation was performed using gradient elution according to Table 1.

[0127] Table 1. Gradient elution conditions

[0128] Time (min) Flow rate (mL / min) Solvent A(%) Solvent B (%) curve <![CDATA[ 0.00 ]]> <![CDATA[ 0.300 ]]> <![CDATA[ 97.0 ]]> <![CDATA[ 3.0 ]]> initial 25.00 0.300 45.0 55.0 6 27.00 0.300 5.0 95.0 6 27.10 0.300 5.0 95.0 6 30.00 0.300 97.0 3.0 6

[0129] An exemplary reference chromatogram from an injection of undigested (intact) mRNA (firefly luciferase; non-lipid encapsulated) is shown in Fig. 7A An exemplary reference chromatogram from an injection of a positive control (RNase T1 digestion of a standard of the same mRNA in solution) is shown in Figure 7B Reference Figure 7B , the peak corresponding to the isolated mRNA digestion product is clearly visible, and the intensity of the peak at a retention time of about 17.5 minutes (corresponding to intact mRNA) is greatly reduced. The chromatogram from the disruption / digestion solution is shown in FIG. Figure 7C Reference Figure 7C, although some digestion may have occurred, no significant signal was observed for the digestion components.

[0130] Example 3. On-adsorbent nuclease digestion of mRNA formulated with lipid nanoparticles (LNPs)

[0131] The firefly luciferase transcript formulated in lipid nanoparticles (LNP-mRNA) was digested on a solid phase extraction (SPE) anion exchange sorbent. The anion exchange sorbent used was a pores and 1.4 μmol / m 2 N,N-bis[3-(methylamino)propyl]methylamine silane bonded 20 μm fumed silica (Daisogel USA, Torrance, CA). Digestion was performed in a 96-well plate format with each well filled with 4 mg of the adsorbent bed. The adsorbent was conditioned with methanol followed by 2% formic acid in water.

[0132] LNP-mRNA preparation (about 100 μ g) is destroyed with the alcohol solution (triethylammonium acetate and n-propanol) containing amine. The preparation destroyed is introduced into the hole containing adsorbent material, and then endonuclease (RNase T1; Excessive, about 30 kU for 10 μg mRNA) is added. Adsorbent plate is covered and placed in 37 ° C baking oven for 30 minutes. 10mM Tris, 0.1mM EDTA, pH 7.5 of 40 μl are used to elute digestion components from adsorbent, and then pH 10 destruction buffer solutions of 40 μl are used to elute. Vacuum is applied between two elutions. Eluent is concentrated (about 10x) and reconstructed in 10mM Tris, 0.1mM EDTA, pH 7.5. As described in Example 2, the sample reconstructed is analyzed by IP-RPLC-MS.

[0133] An exemplary reference chromatogram from an injection of a positive control (in-solution RNase T1 digest of a standard of the same mRNA) is shown in Fig. 8A and relative to Fig. 8A The enlarged view of Fig. 9A Reference Fig. 8A and Fig. 9A , the peak corresponding to the separated mRNA digestion product is clearly visible, and the intensity of the peak at a retention time of about 17.9 minutes (corresponding to intact mRNA) is greatly reduced. The chromatogram from the nuclease digested sample on the adsorbent is shown in FIG. Figure 8B provided, and relative to Figure 8B The enlarged view of Fig. 9B Reference Figure 8B and Fig. 9B, it was observed that mRNA in LNP-mRNA formulations was digested on the adsorbent. The intensity of the digestion fraction eluted after digestion on the adsorbent was lower than that in the case of free mRNA digestion (approximately 1 order of magnitude), but the chromatographic characteristics were highly reproducible ( Figure 8B and Fig. 9B These results also demonstrate that the on-adsorbent digestion profile of mRNA from the LNP-mRNA formulations is comparable to the digestion profile of unformulated mRNA (i.e., the method is digesting the mRNA portion of the LNP-mRNA formulations, resulting in the same distribution of digestion products).

Claims

1. A method for preparing a sample for composition analysis, the method comprising: providing a sample comprising a nucleic acid encapsulated by lipid particles; dissolving the sample comprising the lipid particle-encapsulated nucleic acid in an organic solvent to provide a solution comprising dissolved lipids and solubilized nucleic acid; as well as The solubilized nucleic acids were adsorbed onto a weak anion exchange (WAX) solid phase adsorbent.

2. The method of claim 1, wherein the WAX ​​solid phase adsorbent comprises a surface having disposed thereon an anion exchanger residue having a pKa in the range of about 5 to about 11, the anion exchanger residue comprising a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

3. The method of claim 1 or 2, wherein the anion exchanger residue comprises piperazine.

4. The method according to any one of claims 1 to 3, wherein the organic solvent comprises a C1-C4 alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide, or a combination thereof.

5. The method according to any one of claims 1 to 3, wherein the organic solvent comprises n-propanol.

6. The method according to any one of claims 1 to 5, wherein the organic solvent further comprises an additive selected from the group consisting of: an acid, a base, a detergent, a buffer, a salt, and combinations thereof.

7. The method of claim 6, wherein the additive comprises an ionizable cationic base.

8. The method of any one of claims 1 to 7, further comprising removing the dissolved lipids from the WAX ​​solid phase adsorbent, thereby forming a lipid sample that is substantially free of nucleic acids, wherein removing the dissolved lipids comprises passing the dissolved lipids through the WAX ​​solid phase adsorbent.

9. The method of claim 8, wherein removing the dissolved lipids further comprises: passing the organic solvent through the WAX ​​solid phase adsorbent and collecting the organic solvent flowing therethrough to provide one or more eluates; as well as The one or more eluates are combined with the solubilized lipids that have been allowed to pass through the WAX ​​solid phase adsorbent.

10. The method according to any one of claims 1 to 9, further comprising eluting the adsorbed nucleic acid from the WAX ​​solid phase adsorbent to form a nucleic acid sample that is substantially free of lipids.

11. The method of claim 10, wherein eluting comprises flowing an aqueous buffer through the WAX ​​solid phase adsorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX ​​solid phase adsorbent, the aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent.

12. The method of claim 11, wherein the aqueous buffer has a pH of about 8 to about 12.5 or about 8.5 to about 12.

13. The method according to any one of claims 1 to 12, further comprising analyzing one or more of the lipid sample and the nucleic acid sample by high performance liquid chromatography, mass spectrometry, or a combination thereof.

14. The method according to any one of claims 10 to 13, further comprising digesting at least a portion of the nucleic acid present in the nucleic acid sample with a nuclease.

15. The method of any one of claims 10 to 14, further comprising hydrolyzing at least a portion of the nucleic acid present in the nucleic acid sample to form a mixture of constituent residues.

16. The method according to any one of claims 1 to 9, further comprising: digesting at least a portion of the solubilized nucleic acids adsorbed on the adsorbent to form an adsorbed mixture of nucleic acid digestion products, wherein the digestion comprises contacting the adsorbent with a nuclease; as well as The mixture of nucleic acid digestion products is eluted from the WAX ​​solid phase adsorbent to form a nucleic acid digestion sample.

17. The method of claim 16, wherein washing the cells comprises flowing an aqueous buffer through the WAX ​​solid phase adsorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX ​​solid phase adsorbent, and wherein the aqueous buffer comprises up to about 50% by volume of a non-aqueous solvent.

18. The method of any one of claims 11 to 17, wherein the non-aqueous solvent comprises a C1-C4 alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide, or a combination thereof.

19. The method of claim 17 or 18, wherein the aqueous buffer has a pH of about 7 to about 12.5 or about 8 to about 12.

20. The method of any one of claims 16 to 19, further comprising analyzing the nucleic acid digestion sample by high performance liquid chromatography, mass spectrometry, or a combination thereof.

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