WO2025166311A1 - Methods and systems for characterizing duplexed oligonucleotides - Google Patents

Abstract

Described herein are methods for characterizing duplexed oligonucleotides in a sample, utilizing liquid chromatography and mass spectrometry. For example, a sample comprising a duplexed oligonucleotide may be contacted to a liquid chromatography column to separate the duplexed oligonucleotide. The separated oligonucleotide may be contacted to a mass spectrometer coupled to the liquid chromatography column. A mass of the separate oligonucleotide may be determined and analyzed to characterize, identify, and/or purify the duplexed oligonucleotide. For example, the methods described herein may be used to characterize duplexed siRNA molecules, such as modified siRNA molecules used for therapeutic purposes.

Description

METHODS AND SYSTEMS FOR CHARACTERIZING DUPLEXED OLIGONUCLEOTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/627,986, filed on February⁷ 1, 2024, which is incorporated by reference in its entirety⁷.

SEQUENCE LISTING

[0002] This application contains a sequence listing submitted electronically in XML format under the file name “Sequence_Listing.xml”, which is hereby incorporated by reference in its entirety⁷. The sequence listing was created on January 31, 2025 and is 125,735 bytes in size.

TECHNICAL FIELD

[0003] The present disclosure relates to methods including using liquid chromatography and mass spectrometry to characterize duplexed oligonucleotides.

INTRODUCTION

[0004] Therapeutic oligonucleotides, such as small interfering RNA molecules (siRNAs), are a rapidly growing class of therapeutic drugs, with increasingly high demand for the development of analytical methods for unbiased analytical characterization to ensure product quality, patient safety, and process robustness. Mass spectrometry' has recently emerged as a powerful analytical tool for therapeutic siRNAs and oligonucleotides that carry⁷ various chemical modifications on nucleobases and/or the ribose-phosphate backbones. However, there exists a growing need for methods to characterize such oligonucleotides with increasing efficacy, depth, and accuracy. The present disclosure relates to method for characterizing duplexed oligonucleotides using liquid chromatography, such as hydrophilic interaction chromatography, coupled to mass spectrometry as a powerful tool for analyzing duplexed oligonucleotides which may be co-formulated drug substances with broadened therapeutic applications.

SUMMARY

[0005] Provided herein are methods for characterizing duplexed oligonucleotides in a sample.

[0006] In some embodiments, the method may comprise: contacting the sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate the duplexed oligonucleotide; contacting the separated duplexed oligonucleotide to a mass spectrometer coupled to the liquid chromatography column to determine a mass of the separated duplexed oligonucleotide; and analyzing the mass of the separated duplexed oligonucleotide to characterize the duplexed oligonucleotide. In some aspects, the mass spectrometer may comprise an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer. In some aspects, the mass spectrometer may comprise tandem mass spectrometers. In some aspects, a mobile phase of the liquid chromatography column may comprise ammonium acetate and acetonitrile. In some aspects, the mobile phase may comprise about 10 mM to about 20 mM ammonium acetate and about 70% to about 90% acetonitrile. In some aspects, the mobile phase may comprise a pH of about 5.0 to about 6.0. In some aspects, the liquid chromatography column may comprise a hydrophilic interaction liquid chromatography column, a reversed phase liquid chromatography column, an ion exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a strong cation exchange chromatography column, a size exclusion chromatography column, an affinity chromatography column, a hydrophobic interaction chromatography column, a mixed-mode chromatography column, or a combination thereof. In some aspects, the duplexed oligonucleotide may be or comprise a siRNA. In some aspects, the siRNA may contain one or more chemical modifications. In some aspects, the one or more chemical modifications may include incorporation of one or more of: phosphorothioate (PS), 2’-O-methyl (2’-OMe), 2’-methoxyl (2’MOE), 2'-Fluoro (2’-F), or terminal GalNAc3.

[0007] In some embodiments, the method may comprise: contacting the sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate the duplexed oligonucleotide; denaturing the separated duplexed oligonucleotide to form at least two denatured single-stranded components; contacting the at least two denatured components to a mass spectrometer coupled to the liquid chromatography column to determine a mass of each denatured component; and analyzing the mass of each denatured component to characterize the duplexed oligonucleotide. In some aspects, the mass spectrometer may comprise an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer. In some aspects, the mass spectrometer may comprise tandem mass spectrometers. In some aspects, a mobile phase of the liquid chromatography column may comprise ammonium acetate and acetonitrile. In some aspects, the mobile phase may comprise about 10 mM to about 20 mM ammonium acetate and about 70% to about 90% acetonitrile. In some aspects, the mobile phase comprises a pH of about 5.0 to about 6.0. In some aspects, the liquid chromatography column may comprise a hydrophilic interaction liquid chromatography column, a reversed phase liquid chromatography column, an ion exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a strong cation exchange chromatography column, a size exclusion chromatography column, an affinity chromatography column, a hydrophobic interaction chromatography column, a mixed-mode chromatography column, or a combination thereof. In some aspects, the at least one duplexed oligonucleotide in the mixture may be or comprise an siRNA. In some aspects, the siRNA may contain one or more chemical modifications. In some aspects, the one or more chemical modifications may include incorporation of one or more of: phosphorothioate (PS), 2’-O- methyl (2’-0Me), 2’-methoxyl (2’MOE), 2'-Fluoro (2’-F), or terminal GalNAc3.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various examples and together with the description, serve to explain the principles of the disclosed examples and embodiments.

[0009] Aspects of the disclosure may be implemented in connection with embodiments illustrated in the attached drawings. These drawings show different aspects of the present disclosure and. where appropriate, reference numerals illustrating like structures, components, materials, and/or elements in different figures are labeled similarly. It is understood that various combinations of the structures, components, and/or elements, other than those specifically shown, are contemplated and are within the scope of the present disclosure.

[0010] Moreover, there are many embodiments described and illustrated herein. The present disclosure is neither limited to any single aspect nor embodiment thereof, nor to any combinations and/or permutations of such aspects and/or embodiments. Moreover, each of the aspects of the present disclosure, and/or embodiments thereof, may be employed alone or in combination with one or more of the other aspects of the present disclosure and/or embodiments thereof. For the sake of brevity, certain permutations and combinations are not discussed and/or illustrated separately herein. Notably, an embodiment or implementation described herein as ‘"exemplary" is not to be construed as preferred or advantageous, for example, over other embodiments or implementations; rather, it is intended reflect or indicate the embodiment(s) is/are “example” embodiment(s).

[0011] FIGS. 1 A-1B are line graphs depicting results of an exemplary method for characterizing duplexed oligonucleotides, according to aspects of the present disclosure. FIG. 1A depicts retention time from HILIC for four exemplary oligonucleotides listed in Table 1, and FIG. IB depicts retention from HILIC for three exemplary oligonucleotides listed in Table 1.

[0012] FIGS. 2A-2B are line graphs depicting results of an exemplary method for characterizing unmodified and modified duplexed oligonucleotides, according to aspects of the present disclosure. FIG. 2A depicts retention time from HILIC of exemplary⁷ oligonucleotides listed in Table 2. FIG. 2B depicts the retention times of FIG. 2A at a 20x zoomed-in view. FIG. 2C depicts HILIC-UV retention factor for the exemplary⁷ oligonucleotides listed in Table 2.

[0013] FIGS. 3A-3B are HILIC-ESLMS traces depicting results of an exemplary⁷ method for characterizing a modified duplexed oligonucleotide (siRNA G2), according to aspects of the present disclosure. FIG. 3 A depicts a trace under non-denaturing condition. FIG. 3B depicts a trace under denaturing conditions.

[0014] FIGS. 4A-4B are IPRP-LC-ESI-MS traces depicting results of an exemplary method for characterizing a modified duplexed oligonucleotide (siRNA G2), according to aspects of the present disclosure. FIG. 4A depicts a trace under non-denaturing condition. FIG. 4B depicts a trace under denaturing conditions.

[0015] FIGS. 5A-5C are HILIC-MS traces depicting results of an exemplary method for characterizing a modified duplexed oligonucleotide (siRNA G2), according to aspects of the present disclosure. FIG. 5A depicts a HILIC-MS trace. FIG. 5B depicts a zoomed-in view of FIG. 5A. FIG. 5C depicts a zoomed-in view of FIG. 5B.

[0016] FIGS. 6A-6D are graphs depicting results of an exemplary method for characterizing a mixture of duplexed oligonucleotide (siRNA G1 and siRNA G2), according to aspects of the present disclosure. FIG. 6A depicts a mass spectrum of the mixture of siRNA G1 and siRNA G2. FIG. 6B depicts a total ion chromatogram of the mixture of siRNA G1 and siRNA G2. FIG. 6C depicts an extracted ion chromatogram for siRNA Gl. FIG. 6D depicts an extracted ion chromatogram for siRNA G2.

[0017] Again, there are many embodiments described and illustrated herein. The present disclosure is neither limited to any single aspect nor embodiment thereof, nor to any combinations and/or permutations of such aspects and/or embodiments. Each of the aspects of the present disclosure, and/or embodiments thereof, may be employed alone or in combination with one or more of the other aspects of the present disclosure and/or embodiments thereof. For the sake of brevity⁷, many of those combinations and permutations are not discussed separately herein. [0018] Notably, for simplicity and clarity of illustration, certain aspects of the figures depict the general structure and/or manner of construction of the various embodiments. Descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring other features. Elements in the figures are not necessarily drawn to scale; the dimensions of some features may be exaggerated relative to other elements to improve understanding of the example embodiments.

DETAILED DESCRIPTION

[0019] Therapeutic oligonucleotides are a rapidly growing class of therapeutic drugs which have resulted in a high demand for the development of analytical methods for unbiased analytical characterization to ensure product quality, patient safety, and process robustness. RNA interference (RNAi) is a method for reducing expression of a target gene through the binding by small single- or double-stranded RNA molecules. Small interfering RNAs (siRNAs) are either double-stranded or single-stranded molecules which are an integral part in the RNA degradation pathway. Small interfering RNAs are becoming a promising class of therapeutics in the treatment of viral infections, cancer and genetic disorders.

[0020] Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is currently the most widely used analytical tool in the analysis of siRNAs. However, several drawbacks exist when implementing IP-RPLC, such as the need for proper ion-pairing reagents to interact accordingly with the siRNA for proper separation. Mass spectrometry (MS) has recently emerged as a powerful analytical tool for therapeutic siRNAs and oligonucleotides that carryvarious chemical modifications on nucleobases and/or the ribose-phosphate backbones.

[0021] This disclosure provides methods to satisfy the aforementioned demands by providing methods to characterize siRNAs by comparing IP-RPLC and hydrophilic interaction chromatography coupled to mass spectrometry (HILIC-MS), in comparison with using IP- RPLC. The methods of the present disclosure provides a robust method for siRNA characterization.

[0022] Unless described otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, particular methods and materials are now described.

[0023] The term “a” should be understood to mean “at least one’' and the terms “about” and “approximately” should be understood to permit standard variation as would be understood by those of ordinary skill in the art, and where ranges are provided, endpoints are included. As used herein, the terms “include,’' “includes,’' and “including” are meant to be non-limiting and are understood to mean “comprise,” “comprises,” and “comprising” respectively. [0024] As used herein, the term “sample” refers to a mixture of molecules that comprises at least an oligonucleotide, such as an siRNA oligonucleotide, that is subjected to manipulation in accordance with the methods of the invention, including, for example, separating, analyzing, extracting, concentrating, profiling and the like.

[0025] As used herein, the terms “nucleic acid”, “oligonucleotide”, “polynucleotide” are used interchangeably. They refer to either naturally-occurring or synthetic polymeric forms of nucleotides. The oligonucleotides of the present invention may be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecules. RNA is a string of ribonucleotides, and DNA is a string of deoxyribonucleotides. DNA and RNA can be single-stranded or double-stranded. Messenger RNA (mRNA) is a single stranded molecule which encodes for the specific amino acid sequence of one or more polypeptide chains. During protein synthesis, the nucleic acid sequence of an mRNA is translated to a sequence of amino acid residues for a protein. A person skilled in the art will understand the four nucleotide monomers (bases, nucleotides) of DNA are adenine (A), cytosine (C), guanine (G), and thymine (T), and will understand that the four nucleotide monomers of RNA are A, C, G and uracil (U), and would further understand a uracil of a RNA sequence would be replaced with thymine in a DNA sequence. [0026] As used herein, the terms “small interfering RNA”, “short interfering RNA”, and “siRNA” refer to a class of double-stranded RNA molecules that can interfere with regulation of gene expression. Typically, siRNA strands are about 20-24 base pairs in length. Modifications to the siRNA can occur. These modifications may be based on the component of the nucleotide modified, such as the base, ribose, or phosphate. Non-limiting examples of siRNA modifications include phosphorothioate (PS), 2’-O-methyl (2’-OMe). 2’-methoxyl (2’MOE), 2’-Fluoro (2’-F), terminal GalNAc3, or locked nucleic acids (LNA). Each siRNA molecule may comprise a sense strand and an antisense strand. The sense strand is complementary' to or mostly complementary' to the antisense strand. The nucleic acid sequence of the sense strand may be the same or highly similar to the nucleic acid sequence of the target mRNA sequence. The antisense strand is complementary' or mostly complementary' to a target mRNA sequence, as well as complementary to or mostly complementary' to the sense strand. The antisense strand may reduce expression of the target mRNA sequence.

[0027] As used herein, the terms “complementary” and “complementarity” refer to a nucleic acid that forms hydrogen bonds with another nucleic acid. For example, a nucleic acid (base. nucleotide) may be complementary' to another nucleic acid (base, nucleotide), such as guanine and cytosine; adenine and thyme; or adenine and uracil. A sequence of nucleic acids may be complementary to another sequence of nucleic acids, following the bonding pairs of G-C, A-T, and A-U.

[0028] As used herein, the terms “denaturing” and “denaturation” refer to a process in which a duplexed oligonucleotide is transformed into two complimentary oligonucleotides. Duplex denaturation can be carried out using a denaturing agent. Non-limiting examples of a denaturing agent include heat, high pH, low pH, reducing agents like Tris(2- carbox ethyl iphosphine (TCEP), or exposure to chaotropic agents. Several chaotropic agents can be used as denaturing agents. Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N- lauroylsarcosine. urea, and salts thereof.

[0029] As used herein, the terms “liquid chromatography” or “LC” refer to a process in which a biological and/or chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the components as they flow through (or into) a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reverse phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. In some aspects, the sample or eluate can be subjected to any one of the aforementioned chromatographic methods or a combination thereof.

[0030] In some exemplary embodiments, the chromatography can be hydrophilic interaction chromatography.

[0031] The term “hydrophilic interaction chromatography” or HILIC is intended to include a process employing a hydrophilic stationary phase and a hydrophobic organic mobile phase in which hydrophilic compounds are retained longer than hydrophobic compounds. In certain embodiments, the process utilizes a water-miscible solvent mobile phase.

[0032] In an exemplary embodiment, the separation was carried out using a mobile phase comprised of acetonitrile (ACN) and ammonium acetate. In a specific aspect, the solvent comprises about 20% ACN, about 30% ACN. about 40% ACN, about 50% ACN, about 60% ACN, about 70% ACN, about 80% ACN, or about 90% ACN including any and all values in between. In a specific aspect, the solvent comprises about 10 mM ammonium acetate, about 11 mM ammonium acetate, about 12 mM ammonium acetate, about 13 mM ammonium acetate, about 14 mM ammonium acetate, about 15 mM ammonium acetate, about 16 mM ammonium acetate, about 17 mM ammonium acetate, about 18 mM ammonium acetate, about 19 mM ammonium acetate or about 20 mM ammonium acetate, including any and all values in between.

[0033] In an exemplary embodiment, the column temperature can be about 35 °C, about 36°C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, or about 45 °C.

[0034] As used herein, the term “mass spectrometer” includes a device capable of identifying specific molecular species and measuring their accurate masses. The term is meant to include any molecular detector into which a polypeptide or peptide may be characterized. A mass spectrometer can include three major parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (as in electrospray ionization) or through separate processes. The choice of ion source depends on the application.

[0035] The mass spectrometer in the methods or systems of the present application may comprise or be, for example, an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer, wherein the mass spectrometer can be coupled to a liquid chromatography system, wherein the mass spectrometer is capable of performing liquid chromatography-mass spectrometry' (LC-MS) analysis or liquid chromatography -parallel reaction monitoring-mass spectrometry (LC- PRM-MS) analysis. In some exemplary embodiments, the identification of peptides is performed using PRM-MS.

[0036] In some exemplary' embodiments, the mass spectrometer may comprise or be a tandem mass spectrometer. As used herein, the term “tandem mass spectrometry ” includes a technique where structural information on sample molecules is obtained by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules be transformed into a gas phase and ionized so that fragments are formed in a predictable and controllable fashion after the first mass selection step. MS/MS, or MS², can be performed by first selecting and isolating a precursor ion (MS¹), and fragmenting it to obtain meaningful information. Tandem MS has been successfully performed with a wide variety of analyzer combinations. Which analyzers to combine for a certain application can be determined by many different factors, such as sensitivity, selectivity, and speed, but also size, cost, and availability. The two major categories of tandem MS methods are tandem-in-space and tandem-in-time, but there are also hybrids where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in-space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m/z separation functions can be designed so that in one section of the instrument ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m/z separation and data acquisition. In tandem-in-time, mass spectrometer ions produced in the ion source can be trapped, isolated, fragmented, and m/z separated in the same physical device.

[0037] As used herein, the term “electrospray ionization” or “ESI” refers to the process of spray ionization in which either cations or anions in solution are transferred to the gas phase via formation and desolvation at atmospheric pressure of a stream of highly charged droplets that result from applying a potential difference between the tip of the electrospray needle containing the solution and a counter electrode. There are generally three major steps in the production of gas-phase ions from electrolyte ions in solution. These include: (a) production of charged droplets at the ES infusion tip; (b) shrinkage of charged droplets by solvent evaporation and repeated droplet disintegrations leading to small highly charged droplets capable of producing gas-phase ions; and (c) the mechanism by w hich gas-phase ions are produced from very small and highly charged droplets. Stages (a)-(c) generally occur in the atmospheric pressure region of the apparatus.

[0038] In some exemplary⁷ aspects, the mass spectrometer can use nanoelectrospray or nanospray ionization. The term “nanoelectrospray” or “nanospray” as used herein refers to electrospray ionization at a very low solvent flow rate, typically hundreds of nanoliters per minute of sample solution or lower, often without the use of an external solvent delivery. The electrospray infusion setup forming a nanoelectrospray can use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs a continuous analysis of small sample (analyte) solution volumes over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary⁷ column and a solvent delivery system to perform chromatographic separations on mixtures prior to analysis by the mass spectrometer.

[0039] As used herein, the term “database” refers to a compiled collection of protein sequences that may⁷ possibly exist in a sample, for example in the form of a file in a FASTA format. Relevant protein sequences may be derived from cDNA sequences of a species being studied. Public databases that may be used to search for relevant protein sequences included databases hosted by, for example, Uniprot or Swiss-prot. Databases may be searched using what are herein referred to as ‘■bioinformatics tools’’. Bioinformatics tools provide the capacity to search uninterpreted MS/MS spectra against all possible sequences in the database(s), and provide interpreted (annotated) MS/MS spectra as an output. Non-limiting examples of such tools are Mascot (matrixscience.com), Spectrum Mill (chem.agilent.com), PLGS (waters.com). PEAKS (bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com/proteinpilot), Phenyx (phenyx-ms.com). Sorcerer (sagenresearch.com), OMSSA (pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem (thegpm.org/TANDEM/), Protein Prospector (prospector.ucsf.edu/prospector/mshome.htm), Byonic (proteinmetrics.com/products/byonic), Sequest (fields.scripps.edu/sequest), Protein Metrics PMI-Intact or Thermo BioPharma Finder.

[0040] It is understood that the present invention is not limited to any of the aforesaid nucleic acid(s), chromatographic method(s), mass spectrometer(s), database(s), bioinformatics tool(s), pH range(s) or value(s), temperature(s), or concentration(s), and any nucleic acid(s), chromatographic method(s). mass spectrometer(s), database(s). bioinformatics tool(s). pH. temperature(s), or concentration(s) can be selected by any suitable means.

[0041] Provided herein are methods for characterizing a duplexed oligonucleotide or a mixture of duplexed oligonucleotides in a sample.

[0042] In some embodiments, a method for characterizing a duplexed oligonucleotide in a sample may comprise the steps of contacting the sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate the duplexed oligonucleotide; contacting the separated duplexed oligonucleotide to a mass spectrometer coupled to the liquid chromatography column to determine a mass of the separated duplexed oligonucleotide; and analyzing the mass of the separated duplexed oligonucleotide to characterize the duplexed oligonucleotide.

[0043] The liquid chromatography column of the method may comprise a mobile phase. A mobile phase of the liquid chromatography column may comprise various solutes and solvents. For example, the mobile phase may comprise ammonium acetate and acetonitrile (ACN). In some embodiments, the mobile phase may comprise about 10 mM to about 20 mM ammonium acetate, about 10 mM ammonium acetate, about 11 mM ammonium acetate, about 12 mM ammonium acetate, about 13 mM ammonium acetate, about 14 mM ammonium acetate, about 15 mM ammonium acetate, about 16 mM ammonium acetate, about 17 mM ammonium acetate, about 18 mM ammonium acetate, about 19 mM ammonium acetate, or about 20 mM ammonium acetate, including any and all values in between. In some embodiments, the mobile phase may comprise about 20% to about 90% ACN, about 20% ACN, about 30% ACN, about 40% ACN, about 50% ACN, about 60% ACN, about 70% ACN, about 80% ACN, or about 90% ACN, including any and all values in between. In some embodiments, the mobile phase may comprise about 10 mM to about 20 mM ammonium acetate and about 70% to about 90% acetonitrile. The mobile phase may comprise a pH value. In some embodiments, the mobile phase comprises a pH of about 5.0 to about 6.0, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.0, including any and all values in between.

[0044] The liquid chromatography column may comprise a temperature. In some embodiments, the column temperature may be about 35 °C to about 45 °C, about 35 °C, about 36°C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, or about 45 °C, including any and all values in between.

[0045] The liquid chromatography column may be or comprise any of the various ty pes of liquid chromatography described herein. For example, the liquid chromatography column may comprise a hydrophilic interaction liquid chromatography column, a reversed phase liquid chromatography column, an ion exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a strong cation exchange chromatography column, a size exclusion chromatography column, an affinity chromatography column, a hydrophobic interaction chromatography column, a mixed-mode chromatography column, or a combination thereof.

[0046] The mass spectrometer of the method may comprise an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer. Alternatively or in addition to, the mass spectrometer may comprise a tandem mass spectrometer.

[0047] The duplexed oligonucleotide may⁷ be or comprise any of the various types of oligonucleotides described herein. For example, the duplexed oligonucleotide may be a double-stranded DNA. a double-stranded RNA, or a siRNA. In some embodiments, the duplexed oligonucleotide is a siRNA.

[0048] The duplexed oligonucleotide may⁷ comprise or contain one or more chemical modifications as described herein. For example, the duplexed oligonucleotide may contain one or more chemical modifications including incorporation of any of phosphorothioate (PS), 2^?-O-methyl (2 -OMe), 2’-methoxyl (2’MOE), 2’-Fluoro (2'-F), terminal GalNAc3, or locked nucleic acids (LNA). In some embodiments, the one or more chemical modifications include incorporation of one or more of: phosphorothioate (PS), 2’-O-methyl (2’-0Me), 2‘- methoxyl (2’MOE), 2'-Fluoro (2'-F). or terminal GalNAc3.

[0049] The methods may further comprise a denaturation step after separating the duplexed oligonucleotide.

[0050] In some embodiments, a method for characterizing a duplexed oligonucleotide in a sample may comprise the steps of contacting the sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate the duplexed oligonucleotide; denaturing the separated duplexed oligonucleotide to form at least two denatured singlestranded components; contacting the at least two denatured components to a mass spectrometer coupled to the liquid chromatography column to determine a mass of each denatured component; and analyzing the mass of each denatured component to characterize the duplexed oligonucleotide.

[0051] The liquid chromatography column of the method may comprise a mobile phase. A mobile phase of the liquid chromatography column may comprise various solutes and solvents. For example, the mobile phase may comprise ammonium acetate and acetonitrile (ACN). In some embodiments, the mobile phase may comprise about 10 mM to about 20 mM ammonium acetate, about 10 mM ammonium acetate, about 11 mM ammonium acetate, about 12 mM ammonium acetate, about 13 mM ammonium acetate, about 14 mM ammonium acetate, about 15 mM ammonium acetate, about 16 mM ammonium acetate, about 17 mM ammonium acetate, about 18 mM ammonium acetate, about 19 mM ammonium acetate, or about 20 mM ammonium acetate, including any and all values in between. In some embodiments, the mobile phase may comprise about 20% to about 90% ACN, about 20% ACN, about 30% ACN, about 40% ACN, about 50% ACN, about 60% ACN, about 70% ACN, about 80% ACN, or about 90% ACN, including any and all values in between. In some embodiments, the mobile phase may comprise about 10 mM to about 20 mM ammonium acetate and about 70% to about 90% acetonitrile. The mobile phase may comprise a pH value. In some embodiments, the mobile phase comprises a pH of about 5.0 to about 6.0, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5. about 5.6, about 5.7, about 5.8, about 5.9. or about 6.0. including any and all values in between.

[0052] The liquid chromatography column may comprise a temperature. In some embodiments, the column temperature may be about 35 °C to about 45 °C, about 35 °C, about 36°C, about 37 °C, about 38 °C. about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, or about 45 °C, including any and all values in between. [0053] The liquid chromatography column may be or comprise any of the various types of liquid chromatography described herein. For example, the liquid chromatography column may comprise a hydrophilic interaction liquid chromatography column, a reversed phase liquid chromatography column, an ion exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a strong cation exchange chromatography column, a size exclusion chromatography column, an affinity chromatography column, a hydrophobic interaction chromatography column, a mixed-mode chromatography column, or a combination thereof.

[0054] Denaturing may comprise contacting the separated duplexed oligonucleotide to a denaturing agent and/or denaturing conditions. In some embodiments, the denaturing agent and/or denaturing conditions may comprise or be heat, high pH, low pH, a reducing agent, and/or a chaotropic agent. In some embodiments, the reducing agent may be or comprise Tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the chaotropic agent may be or comprise butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroylsarcosine, urea, or salts thereof.

[0055] The mass spectrometer of the method may comprise an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer. Alternatively or in addition to, the mass spectrometer may comprise a tandem mass spectrometer.

[0056] The duplexed oligonucleotide may be or comprise any of the various types of oligonucleotides described herein. For example, the duplexed oligonucleotide may be a double-stranded DNA, a double-stranded RNA, or a siRNA. In some embodiments, the duplexed oligonucleotide is a siRNA.

[0057] The duplexed oligonucleotide may comprise or contain one or more chemical modifications as described herein. For example, the duplexed oligonucleotide may contain one or more chemical modifications including incorporation of any of phosphorothioate (PS), 2’-O-methyl (2’-OMe), 2’-methoxyl (2'MOE), 2'-Fluoro (2’-F), terminal GalNAc3, or locked nucleic acids (LNA). In some embodiments, the one or more chemical modifications include incorporation of one or more of: phosphorothioate (PS), 2’-O-methyl (2’-0Me), 2’- methoxyl (2’MOE), 2'-Fluoro (2’-F), or terminal GalNAc3.

[0058] The present disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. EXAMPLES

[0059] The present disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. [0060] Materials. Duplexed siRNAs and the corresponding single-stranded RNA oligonucleotide sense (S) and anti-sense (AS) components were purchased from IDT or synthesized in-house. In addition to partial backbone modification with phosphorothioate (PS), common chemical modifications such as deoxyribonucleic acid (DNA), 2'-Fluoro (2’- F), 2'-O-methoxy-ethyl (2’-M0E), and terminal GalNAc3 were selectively incorporated into non-targeting siRNA sequences.

[0061] UPLC separation using Hydrophilic Interaction Liquid Chromatography

(HILIC). The mixtures containing siRNA duplex and impurities were subjected to UPLC separation. HILIC-MS experiments were conducted using several columns, including Waters ACQUITY Premier BEH Amide Column (1.7 pm, 2.1 x 150 mm with the column compartment set to 40 °C), Agilent InfinityLab Poroshell 120 HILIC-Z Column, and others. Mobile phase A was comprised of 15 rnM ammonium acetate (pH 5.5) in 80% acetonitrile, and mobile phase B was comprised of 15 rnM ammonium acetate (pH 5.5) in 20% acetonitrile. A gradient flow of 20% to 60% mobile phase B in 10 minutes was used at a flow rate of 0.25 mL/min. The eluent was monitored at 260 nm by photo diode array (PDA) detector and introduced to MS analysis by heated electrospray ionization (ESI) in negative ion mode. All LC-MS/MS experiments were performed on a Waters ACQUITY™ UPLC I- Class System coupled with a Q EXACTIVE™ Plus Hybrid QUADRUPOLE-ORBITRAP™ Mass Spectrometer. Intact mass analysis was performed using Protein Metrics PMI-Intact (v5.3) or Thermo BioPharma Finder (v5. 1).

[0062] UPLC separation using non-denaturing ion-pairing reversed-phase liquid chromatography. The mixtures containing siRNA duplex and impurities were subjected to UPLC separation. Non-denaturing ion-pairing reversed-phase liquid chromatography (ndlP- RPLC) was performed using Waters ACQUITY Premier Oligonucleotide BEH C18 Column, 300 A, 1.7 pm, 2. 1 x 100 mm with the column temperature at 25 °C. Mobile phase A was comprised of 0.2% triethylamine (TEA) and 0.5% l,LL3,3,3-hexafluoro-2-propanol (HFIP) solution (v/v) in Milli-Q water and mobile phase B was comprised of 80% methanol and 20% acetonitrile. A gradient flow of 0% B to 50% B in 20 min was used at a flow rate of 0.25 mL/min. The eluent was monitored at 260 nm by PDA detector and introduced to MS analysis by heated electrospray ionization (ESI) in negative ion mode. All LC-MS/MS experiments were performed on a Waters ACQUITY™ UPLC I-Class System coupled with a Q EXACTIVE™ Plus Hybrid QUADRUPOLE-ORBITRAP™ Mass Spectrometer. Intact mass analysis was performed using Protein Metrics PMI -Intact (v5.3) or Thermo BioPharma Finder (v5. 1).

[0063] Example 1: HILIC Retention of siRNA Oligonucleotides

[0064] To assess the impact of chemical modifications on small interfering RNA (siRNA) oligonucleotides. HILIC-MS was carried out on synthetic duplexed and single stranded siRNA molecules. Table I, below, lists the different modifications utilized on the synthetic siRNA molecules.

Table 1

[0065] The effects of buffer salts (ammonium acetate, ammonium formate), ionic strength, column temperature, and types of HILIC columns were investigated. Single or mixtures of siRNAs were injected at a concentration of 10 pmol/pL and separated by HILIC using a 10- min gradient followed by 5-min column equilibration. Mobile phases were buffered with 15 mM ammonium acetate (pH 5.5) with water as a strong elution solvent and acetonitrile as a weak solvent. Full-scan MS spectra were acquired on a mass spectrometer operated in negative ion mode using mass resolution of 120,000 to achieve isotopic resolution. FIGS. 1A- 1B show retention time variation for each chemical modification.

[0066] To further characterize the HILIC retention time and the effects of chemical modifications, additional siRNA sense and antisense sequences were tested, as shown in Table 2, below.

Table 2

[0067] As shown in FIGS. 2A-2B, the modified siRNA oligonucleotides migrated faster than the unmodified siRNA oligonucleotides. The retention factor was then calculated for the various duplex siRNAs as shown in FIG. 2C. Overall, HILIC and mixed mode HILIC demonstrated attractive performance in separating duplexed siRNAs and their single-strand S/AS components with complete absence of ion-paring reagents. This method resulted in diminished carryover between injections, shorter chromatographic gradient and higher LC peak capacity.

[0068] In addition, HILIC-ESI-MS largely preserved the duplex conformation of siRNAs, with major charge envelopes ranging from [M-5H]⁵' to [M-7H]⁷', as shown in FIG. 3 A. Using HILIC with denaturing ESI-MS. dissociation of the duplex to the sense and anti-sense strands was observed, as shown in FIG. 3B. These findings were compared with traditional nondenaturing ion-pairing reverse phase liquid chromatography -mass spectrometry (IP-RP LC- MS) results. As shown in FIG. 4A, siRNA duplexes remained intact under chromatographic separation with major charge envelopes ranging from [M-9H] ⁹‘ to [M-13H] ^13-. Dissociation into sense (S) and anti-sense (AS) strands in the gas phase during ESI was also observed, as shown in FIG. 4B. [0069] Mass deconvolution was successfully applied to the main duplex peak(s) as well as single-stranded impurities, as shown in FIG. 5A-5C. Intact mass analysis was performed by using Protein Metrics PMI-Intact (v5.3) or Thermo BioPharma Finder (v5.1).

[0070] Example 2: Characterization and Quantification of siRNA Components

[0071] The methods described herein were shown to successfully differentiate between two duplexed siRNAs in a mixture. A one-to-one (1 :1) mixture of siRNA G1 and siRNA G2 was prepared and evaluated using the methods described above.

[0072] The ESI-MS trace shown in FIG. 6A demonstrated the duplex ion formed of both siRNA G1 and siRNA G2 at [M-6H]⁶’ and [M-7J]⁷’.

[0073] FIGS. 6B-6D show- the total ion chromatogram (FIG. 6B) and the extracted ion chromatograms (FIG. 6C and FIG. 6D) for duplex siRNA G1 and siRNA G2. The extracted ion chromatograms for duplex siRNA G1 and siRNA G2 showed similar transition peak intensity for the ion peak present at [M-6H]⁶'. For quantification of each siRNA component in the mixture, the total area under the curve (AUC) for each extracted charge state and the calculated AUC from deconvoluted mass were used.

[0074] The developed HILIC-MS approach represents an attractive and powerful tool for analyzing individual siRNA in a mixture or characterizing co-formulated drug substances with broadened therapeutic applications.

[0075] The present disclosure can be further understood in the embodiments of the following items.

[0076] Item 1 . A method for isolating and/or purifying duplexed oligonucleotide in a sample, comprising: contacting a sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate duplexed oligonucleotides to form a separated sample; contacting the separated sample to a mass spectrometer that is coupled to the liquid chromatography column to determine a mass of the separated duplexed oligonucleotide material; and analyzing the mass of the duplexed oligonucleotide material to isolate and/or purify the duplexed oligonucleotide.

[0077] Item 2. The method of item 1, wherein said oligonucleotide is siRNA.

[0078] Item 3. The method of item 1, wherein said liquid chromatography step comprises reversed phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, weak cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction liquid chromatography (HILIC), mixedmode chromatography, or a combination thereof.

[0079] Item 4. The method of item 2, wherein said liquid chromatography step comprises HILIC.

[0080] Item 5. The method of item 1, said mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer, wherein said mass spectrometer is coupled to said liquid chromatography system.

[0081] Item 6. The method of item 1, wherein said mass spectrometry is tandem mass spectrometry.

[0082] Item 7. The method of item 1, wherein the mobile phase of the liquid chromatography comprises ammonium acetate in acetonitrile (ACN).

[0083] Item 8. The method of item 7, wherein the concentration of ammonium acetate is from about 10 mM ammonium acetate to about 20 mM ammonium acetate and the amount of ACN is about 70% ACN to about 90% ACN.

[0084] Item 9. The method of item 8, wherein the mobile phase comprises about 15 mM ammonium acetate in about 80% ACN.

[0085] Item 10. The method of item 7, wherein the pH of the mobile phase is about pH 5.0 to about pH 6.0.

[0086] Item 11. The method of item 9, wherein the pH of the mobile phase is about pH 5.5.

[0087] Item 12. The method of item 2, wherein the siRNA contains one or more chemical modifications.

[0088] Item 13. The method of item 13, wherein the chemical modifications include the incorporation of one or more of the following to the siRNA: phosphorothioate (PS), 2’-O- methyl (2’-OMe), 2’-methoxyl (2’MOE), 2'-Fluoro (2’-F), terminal GalNAc3, or a combination thereof.

[0089] Item 14. A method for isolating and/or purifying duplexed oligonucleotide in a sample, comprising: contacting a sample comprising the duplexed oligonucleotide to hydrophilic interaction liquid chromatography (HILIC) to separate duplexed oligonucleotides to form a separated sample; contacting the separated sample to a mass spectrometer that is coupled to the liquid chromatography column to determine a mass of the duplexed oligonucleotide; and analyzing the mass of the duplexed oligonucleotide material to isolate and/or purify the duplexed oligonucleotide.

[0090] Item 15. The method of item 1, wherein said oligonucleotide is siRNA.

[0091] Item 16. The method of item 14, said mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer, wherein said mass spectrometer is coupled to said liquid chromatography system.

[0092] Item 17. The method of item 14, wherein said mass spectrometry is tandem mass spectrometry.

[0093] Item 18. The method of item 14, wherein the mobile phase of the liquid chromatography comprises ammonium acetate in acetonitrile (ACN).

[0094] Item 19. The method of item 18, wherein the concentration of ammonium acetate is from about 10 mM ammonium acetate to about 20 mM ammonium acetate and the amount of ACN is about 70% ACN to about 90% ACN.

[0095] Item 20. The method of item 19, wherein the mobile phase comprises about 15 mM ammonium acetate in about 80% ACN.

[0096] Item 21. The method of item 18, wherein the pH of the mobile phase is about pH 5.0 to about pH 6.0.

[0097] Item 22. The method of item 21, wherein the pH of the mobile phase is about pH 5.5.

[0098] Item 23. The method of item 15. wherein the siRNA contains one or more chemical modifications.

[0099] Item 24. The method of item 23, wherein the chemical modifications include the incorporation of one or more of the following to the siRNA: phosphorothioate (PS), 2 -O- methyl (2'-OMe). 2’-methoxyl (2’MOE), 2'-Fluoro (2'-F). terminal GalNAc3, or a combination thereof.

[0100] Item 25. A method for characterizing duplexed oligonucleotide in a sample, comprising: contacting a sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate duplexed oligonucleotide; denaturing the separated duplexed sample to generate single stranded components to form a denatured sample; contacting the denatured sample to a mass spectrometer that is coupled to the liquid chromatography column to determine a mass of the single stranded components; and analyzing the mass of the single stranded components to characterize the duplex oligonucleotide.

[0101] Item 26. The method of item 25, wherein said oligonucleotide is siRNA.

[0102] Item 27. The method of item 25, wherein the single stranded components are sense and anti-sense components.

[0103] Item 28. The method of item 25, wherein said liquid chromatography step comprises reversed phase liquid chromatography, ion exchange chromatography, anion exchange chromatography, weak cation exchange chromatography, strong cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction liquid chromatography (HILIC), mixedmode chromatography, or a combination thereof.

[0104] Item 29. The method of item 28, wherein said liquid chromatography step comprises HILIC.

[0105] Item 30. The method of item 25, said mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer, wherein said mass spectrometer is coupled to said liquid chromatography system.

[0106] Item 31. The method of item 25, wherein said mass spectrometry is tandem mass spectrometry.

[0107] Item 32. The method of item 25. wherein the mobile phase of the liquid chromatography comprises ammonium acetate in acetonitrile (ACN).

[0108] Item 33. The method of item 32, wherein the concentration of ammonium acetate is from about 10 mM ammonium acetate to about 20 mM ammonium acetate and the amount of ACN is about 70% ACN to about 90% ACN.

[0109] Item 34. The method of item 33, wherein the mobile phase comprises about 15 mM ammonium acetate in about 80% ACN.

[0110] Item 35. The method of item 32, wherein the pH of the mobile phase is about pH 5.0 to about pH 6.0.

[0111] Item 36. The method of item 34. wherein the pH of the mobile phase is about pH 5.5. [0112] Item 37. The method of item 26, wherein the siRNA contains one or more chemical modifications.

[0113] Item 38. The method of item 37, wherein the chemical modifications include the incorporation of one or more of the following to the siRNA: phosphorothioate (PS), 2’-O- methyl (2’-0Me), 2’-methoxyl (2'MOE), 2'-Fluoro (2’-F), terminal GalNAc3, or a combination thereof.

[0114] Item 39. A method for characterizing duplexed oligonucleotide in a sample, comprising: contacting a sample comprising the duplexed oligonucleotide to hydrophilic interaction liquid chromatography (HILIC) to separate duplexed oligonucleotide to form a separated sample; denaturing the separated sample to generate single stranded components to form a denatured sample; contacting the denatured sample to a mass spectrometer that is coupled to the liquid chromatography column to determine a mass of the single stranded components; and analyzing the mass of the single stranded components to characterize the duplexed oligonucleotide.

[0115] Item 40. The method of item 39, wherein said oligonucleotide is siRNA.

[0116] Item 41. The method of item 39, wherein the single stranded components are sense and anti-sense components.

[0117] Item 42. The method of item 39, said mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer, wherein said mass spectrometer is coupled to said liquid chromatography system.

[0118] Item 43. The method of item 39, wherein said mass spectrometry is tandem mass spectrometry.

[0119] Item 44. The method of item 39, wherein the mobile phase of the liquid chromatography comprises ammonium acetate in acetonitrile (ACN).

[0120] Item 45. The method of item 44, wherein the concentration of ammonium acetate is from about 10 mM ammonium acetate to about 20 mM ammonium acetate and the amount of ACN is about 70% ACN to about 90% ACN.

[0121] Item 46. The method of item 45, wherein the mobile phase comprises about 15 mM ammonium acetate in about 80% ACN.

[0122] Item 47. The method of item 44, wherein the pH of the mobile phase is about pH 5.0 to about pH 6.0.

[0123] Item 48. The method of item 46, wherein the pH of the mobile phase is about pH 5.5. [0124] Item 49. The method of item 40, wherein the siRNA contains one or more chemical modifications. [0125] Item 50. The method of item 49, wherein the chemical modifications include the incorporation of one or more of the following to the siRNA: phosphorothioate (PS), 2’-O- methyl (2’-OMe), 2’-methoxyl (2’MOE), 2'-Fluoro (2’-F), terminal GalNAc3, or a combination thereof.

Claims

1. A method for characterizing a duplexed oligonucleotide in a sample, comprising: contacting the sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate the duplexed oligonucleotide; contacting the separated duplexed oligonucleotide to a mass spectrometer coupled to the liquid chromatography column to determine a mass of the separated duplexed oligonucleotide; and analyzing the mass of the separated duplexed oligonucleotide to characterize the duplexed oligonucleotide.

2. The method of claim 1 , wherein the mass spectrometer comprises an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer.

3. The method of claim 1, wherein the mass spectrometer comprises a tandem mass spectrometer.

4. The method of claim 1, wherein a mobile phase of the liquid chromatography column comprises ammonium acetate and acetonitrile.

5. The method of claim 4, wherein the mobile phase comprises about 10 mM to about 20 mM ammonium acetate and about 70% to about 90% acetonitrile.

6. The method of claim 4, wherein the mobile phase comprises a pH of about 5.0 to about 6.0.

7. The method of claim 1, wherein the liquid chromatography column comprises a hydrophilic interaction liquid chromatography column, a reversed phase liquid chromatography column, an ion exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a strong cation exchange chromatography column, a size exclusion chromatography column, an affinity chromatography column, a hydrophobic interaction chromatography column, a mixed-mode chromatography column, or a combination thereof.

8. The method of claim 1, wherein the duplexed oligonucleotide is an siRNA.

9. The method of claim 8, wherein the siRNA contains one or more chemical modifications.

10. The method of claim 9, wherein the one or more chemical modifications include incorporation of one or more of: phosphorothioate (PS). 2’-O-methyl (2’-OMe), 2’-methoxyl (2’MOE), 2'-Fluoro (2’-F), or terminal GalNAc3.

11. A method for characterizing a duplexed oligonucleotide in a sample, comprising: contacting the sample comprising the duplexed oligonucleotide to a liquid chromatography column to separate the duplexed oligonucleotide; denaturing the separated duplexed oligonucleotide to form at least two denatured single-stranded components; contacting the at least two denatured components to a mass spectrometer coupled to the liquid chromatography column to determine a mass of each denatured component; and analyzing the mass of each denatured component to characterize the duplexed oligonucleotide.

12. The method of claim 1 1 , wherein the mass spectrometer comprises an electrospray ionization mass spectrometer, a nano-electrospray ionization mass spectrometer, or an Orbitrap-based mass spectrometer.

13. The method of claim 11, wherein the mass spectrometer comprises a tandem mass spectrometer.

14. The method of claim 11, wherein a mobile phase of the liquid chromatography column comprises ammonium acetate and acetonitrile.

15. The method of claim 14, wherein the mobile phase comprises about 10 mM to about 20 mM ammonium acetate and about 70% to about 90% acetonitrile.

16. The method of claim 14, wherein the mobile phase comprises a pH of about 5.0 to about 6.0.

17. The method of claim 1, wherein the liquid chromatography column comprises a hydrophilic interaction liquid chromatography column, a reversed phase liquid chromatography column, an ion exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a strong cation exchange chromatography column, a size exclusion chromatography column, an affinity chromatography column, a hydrophobic interaction chromatography column, a mixed-mode chromatography column, or a combination thereof.

18. The method of claim 11, wherein at least one duplexed oligonucleotide in the mixture is an siRNA.

19. The method of claim 18, wherein the siRNA contains one or more chemical modifications.

20. The method of claim 19, wherein the one or more chemical modifications include incorporation of one or more of: phosphorothioate (PS), 2’-O-methyl (2’-OMe), 2'-methoxyl (2’MOE), 2'-Fluoro (2^?-F), or terminal GalNAc3.