Optimization method for single strand purity analysis of a sirna product

Abstract

The present disclosure provides systems and methods for separating and characterizing oligonucleotides. Fixed dose combinations of small interfering RNAs can be subjected to denaturing ion pairing reverse phase liquid chromatography using optimal conditions to separate single RNA strands for purity analysis.

Description

DOCKET NO.: 38120-6046 (11853WO01) OPTIMIZATION METHOD FOR SINGLE STRAND PURITY ANALYSIS OF A SIRNA PRODUCT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent ApplicationNo.63 / 647,402, filed May 14, 2024 which is incorporated herein by reference in its entirety for all purposes. Field

[0002] The present disclosure is directed, in part, to methods and systems for separating andcharacterizing oligonucleotides. These methods and systems are based on denaturing ion pairing reverse phase liquid chromatography analysis. BACKGROUND

[0003] Small interfering RNA (siRNA) is an increasingly important class of therapeuticoligonucleotides. The effectiveness of siRNA therapies can be further improved through the use of fixed dose combinations (FDCs), comprising multiple different siRNAs. However, the combination of siRNAs, which may be highly structurally and physicochemically similar, creates a challenge in separating the siRNAs for analysis when using conventional analytical methods. In particular, a conventional denaturing ion pairing reverse phase liquid chromatography (d- IPRP-LC) method was incapable of sufficiently separating each of the strands of two siRNAs in an FDC.

[0004] It will be appreciated that a need exists for sensitive systems and methods for separating,quantifying, and characterizing siRNAs and strands thereof in fixed dose combinations. SUMMARY

[0005] This disclosure provides methods for separating at least two oligonucleotides in a sample.In some exemplary embodiments, the methods can comprise obtaining a sample comprising at least two oligonucleotides, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotides, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injectionDOCKET NO.: 38120-6046 (11853WO01) volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

[0006] In some embodiments, said at least two oligonucleotides are sense strands of smallinterfering RNAs. In some embodiments, said at least two oligonucleotides are antisense strands of small interfering RNAs.

[0007] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0008] In some embodiments, said at least two oligonucleotides are detected using ultravioletdetection.

[0009] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.

[0010] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0011] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.

[0012] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.

[0013] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0014] This disclosure further provides methods for separating at least two oligonucleotidestrands in a sample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising at least two oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.DOCKET NO.: 38120-6046 (11853WO01)

[0015] In some embodiments, said at least two oligonucleotide strands are sense strands of smallinterfering RNAs. In some embodiments, said at least two oligonucleotide strands are antisense strands of small interfering RNAs.

[0016] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0017] In some embodiments, said at least two oligonucleotide strands are detected usingultraviolet detection.

[0018] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.

[0019] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0020] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.

[0021] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.

[0022] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0023] This disclosure further provides methods for separating a first single-strandedoligonucleotide from at least one additional single-stranded oligonucleotide. In some exemplary embodiments, the methods can comprise obtaining a sample comprising a first single-stranded oligonucleotide and at least one additional single-stranded oligonucleotide, and subjecting said sample to denaturing ion pairing liquid chromatography separation, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

[0024] In some embodiments, said first single-stranded oligonucleotide and said at least oneadditional single-stranded oligonucleotide are sense strands of small interfering RNAs. In someDOCKET NO.: 38120-6046 (11853WO01) embodiments, said first single-stranded oligonucleotide and said at least one additional single- stranded oligonucleotide are antisense strands of small interfering RNAs.

[0025] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0026] In some embodiments, said single-stranded oligonucleotides are detected using ultravioletdetection.

[0027] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.

[0028] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0029] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.

[0030] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.

[0031] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0032] This disclosure further provides methods for separating at least two oligonucleotidestrands in a sample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising at least two oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%.

[0033] In some embodiments, said at least two oligonucleotide strands are antisense strands ofsmall interfering RNAs.

[0034] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.DOCKET NO.: 38120-6046 (11853WO01)

[0035] This disclosure further provides methods for separating four oligonucleotide strands in asample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising four oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%, wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA.

[0036] In some embodiments, each of said two small interfering RNAs comprises anoligonucleotide strand that is connected to a non-oligonucleotide moiety. In some embodiments, said non-oligonucleotide moiety comprises a GalNAc moiety.

[0037] In some embodiments, each of said four oligonucleotide strands comprises aphosphorothioate (PS) backbone modification.

[0038] This disclosure further provides methods for separating four oligonucleotide strands in asample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising four oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%, wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA, wherein each small interfering RNA comprises an oligonucleotide strand that is connected to a non-oligonucleotide moiety that comprises a GalNAc moiety, and wherein each of said four oligonucleotide strands comprises a phosphorothioate (PS) backbone modification.DOCKET NO.: 38120-6046 (11853WO01)

[0039] This disclosure further provides methods for separating four oligonucleotide strands in asample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising four oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of 85 °C, an injection volume of 2 µL, a mobile phase B comprising 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at T=0min an initial concentration of said mobile phase B is 5% and at T=40min a concentration of said mobile phase B is 22%, wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA, wherein each of said two small interfering RNAs comprises an oligonucleotide strand that is connected to a non- oligonucleotide moiety that comprises a GalNAc moiety, and wherein each of said four oligonucleotide strands comprises a phosphorothioate (PS) backbone modification.

[0040] This disclosure provides methods for separating at least two oligonucleotides in a sample.In some exemplary embodiments, the methods can comprise obtaining a sample comprising at least two oligonucleotides, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotides, wherein conditions for said separation include a column temperature from about 75 °C to about 90 °C, an injection volume from about 1 µL to about 8 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 10% and a maximum concentration of said mobile phase B is from about 18% to about 27%.

[0041] In some embodiments, said at least two oligonucleotides are sense strands of smallinterfering RNAs. In some embodiments, said at least two oligonucleotides are antisense strands of small interfering RNAs.

[0042] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0043] In some embodiments, said at least two oligonucleotides are detected using ultravioletdetection.

[0044] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.DOCKET NO.: 38120-6046 (11853WO01)

[0045] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0046] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.

[0047] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.

[0048] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0049] This disclosure further provides methods for separating at least two oligonucleotidestrands in a sample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising at least two oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature from about 75 °C to about 90 °C, an injection volume from about 1 µL to about 8 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 10% and a maximum concentration of said mobile phase B is from about 18% to about 27%.

[0050] In some embodiments, said at least two oligonucleotide strands are sense strands of smallinterfering RNAs. In some embodiments, said at least two oligonucleotide strands are antisense strands of small interfering RNAs.

[0051] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0052] In some embodiments, said at least two oligonucleotide strands are detected usingultraviolet detection.

[0053] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.

[0054] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0055] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.DOCKET NO.: 38120-6046 (11853WO01)

[0056] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.

[0057] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0058] This disclosure further provides methods for separating a first single-strandedoligonucleotide from at least one additional single-stranded oligonucleotide. In some exemplary embodiments, the methods can comprise obtaining a sample comprising a first single-stranded oligonucleotide and at least one additional single-stranded oligonucleotide, and subjecting said sample to denaturing ion pairing liquid chromatography separation, wherein conditions for said separation include a column temperature from about 75 °C to about 90 °C, an injection volume from about 1 µL to about 8 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 10% and a maximum concentration of said mobile phase B is from about 18% to about 27%.

[0059] In some embodiments, said first single-stranded oligonucleotide and said at least oneadditional single-stranded oligonucleotide are sense strands of small interfering RNAs. In some embodiments, said first single-stranded oligonucleotide and said at least one additional single- stranded oligonucleotide are antisense strands of small interfering RNAs.

[0060] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0061] In some embodiments, said single-stranded oligonucleotides are detected using ultravioletdetection.

[0062] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.

[0063] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0064] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.

[0065] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.DOCKET NO.: 38120-6046 (11853WO01)

[0066] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1A shows a denaturing ion pairing reverse phase liquid chromatography (d-IPRP-LC) chromatogram illustrating separation of each strand of two small interfering RNAs (siRNAs) using an established method (i.e., the Base Method described herein).

[0068] FIG. 1B illustrates a mobile phase gradient (“Gradient A”) used in an established d-IPRP-LC method (i.e., the Base Method described herein).

[0069] FIG. 2A shows a d-IPRP-LC chromatogram of a preliminary method assessment ofcritical main factors for d-IPRP-LC separation of siRNA strands using a peptide column.

[0070] FIG. 2B shows an enlarged view of the d-IPRP-LC chromatogram shown in FIG. 2A.

[0071] FIG. 2C shows schematics of mobile phase gradients used for preliminary methodassessments of critical main factors for d-IPRP-LC separation of siRNA strands.

[0072] FIG. 2D shows a d-IPRP-LC chromatogram of a preliminary method assessment ofcritical main factors for d-IPRP-LC separation of siRNA strands using a peptide column.

[0073] FIG. 2E shows a d-IPRP-LC chromatogram of preliminary method assessment of criticalmain factors for d-IPRP-LC separation of siRNA strands using a peptide column.

[0074] FIG. 3A shows a d-IPRP-LC chromatogram of preliminary method assessment of criticalmain factors for d-IPRP-LC separation of siRNAs strands using an oligonucleotide column (except for the Base Method / Gradient A, which used a peptide column); ON Col = Oligonucleotides Column.

[0075] FIG. 3B shows an enlarged view of the d-IPRP-LC chromatogram shown in FIG. 3A.

[0076] FIG. 4 shows a workflow for a Design of Experiments (DoE) study for evaluating d-IPRP-LC method parameters.

[0077] FIG. 5A shows a d-IPRP-LC chromatogram with severe siRNA sense strand peaksplitting (encircled).

[0078] FIG. 5B shows a schematic illustrating normalization of an siRNA sense strand peak in ad-IPRP-LC chromatogram that shows severe peak splitting.DOCKET NO.: 38120-6046 (11853WO01)

[0079] FIG. 5C shows a d-IPRP-LC chromatogram with mild siRNA antisense strand peaksplitting.

[0080] FIG. 5D shows a schematic illustrating normalization of an siRNA antisense strand peakin a d-IPRP-LC chromatogram that shows mild peak splitting.

[0081] FIG. 6A shows actual versus predicted values for siRNA antisense strand resolution in aDoE study of d-IPRP-LC method parameters.

[0082] FIG. 6B shows residual versus predicted values for siRNA antisense strand resolution ina DoE study of d-IPRP-LC method parameters.

[0083] FIG. 6C shows a prediction profiler for siRNA antisense strand resolution in a DoE studyof d-IPRP-LC method parameters.

[0084] FIG. 6D shows scaled estimates for factors affecting siRNA antisense strand resolution ina DoE study of d-IPRP-LC method parameters.

[0085] FIG. 7A shows actual versus predicted values for siRNA sense strand resolution in a DoEstudy of d-IPRP-LC method parameters.

[0086] FIG. 7B shows residual versus predicted values for siRNA sense strand resolution in aDoE study of d-IPRP-LC method parameters.

[0087] FIG. 7C shows a prediction profiler for siRNA sense strand resolution in a DoE study ofd-IPRP-LC method parameters.

[0088] FIG. 7D shows scaled estimates for factors affecting siRNA sense strand resolution in aDoE study of d-IPRP-LC method parameters.

[0089] FIG. 8A shows actual versus predicted values for overall peak shape in a DoE study of d-IPRP-LC method parameters.

[0090] FIG. 8B shows residual versus predicted values for overall peak shape in a DoE study ofd-IPRP-LC method parameters.

[0091] FIG. 8C shows a prediction profiler for overall peak shape in a DoE study of d-IPRP-LCmethod parameters.

[0092] FIG. 8D shows scaled estimates for factors affecting overall peak shape in a DoE study ofd-IPRP-LC method parameters.

[0093] FIG. 9 shows a prediction profiler for various responses in a DoE study of d-IPRP-LCmethod parameters.DOCKET NO.: 38120-6046 (11853WO01)

[0094] FIG. 10A shows d-IPRP-LC characterization of the siRNA sense and antisense strands ofan siRNA-1-siRNA-2 fixed dose combination using an established method (i.e., the Base Method described herein).

[0095] FIG. 10B shows d-IPRP-LC characterization of the siRNA sense and antisense strands ofan siRNA-1-siRNA-2 fixed dose combination using an optimal method determined from DoE modeling.

[0096] FIG. 11 shows siRNA sense and antisense strands separation by d-IPRP-LC of separatesingle siRNA solutions using an optimal method determined from DoE modeling.

[0097] FIG. 12 shows d-IPRP-LC characterization of the siRNA sense and antisense strands ofthree different siRNA fixed dose combinations (FDCs; each including two different siRNAs) using an optimal method determined from DoE modeling. DETAILED DESCRIPTION

[0098] The disclosure is based, in part, on the inventors’ finding that oligonucleotide strandswith highly similar physicochemical properties can be separated and characterized by d-IPRP- LC using specific column temperatures, sample injection volumes, particular mobile phase B compositions, or mobile phase B gradients.

[0099] Small interfering RNA (siRNA) has emerged as a rapidly growing class of therapeuticoligonucleotides, with six siRNA drug approvals since 2018. Compared to monotherapy, fixed dose combinations (FDC) of siRNA can offer potential benefits including simplified administration, enhanced efficacy, reduced resistance, and improved specificity. A major challenge in the analytical development for FDC products of siRNA is accurately quantifying and characterizing each siRNA component within the formulation, particularly when the siRNA components have highly similar physicochemical properties. This disclosure sets forth a Design of Experiment (DoE) approach to optimize a denaturing ion pairing reverse phase liquid chromatography (d-IPRP-LC) method for single strand purity analysis of a siRNA FDC product, and an optimal method.

[0100] The two representative siRNA molecules in the FDC have identical length and highlysimilar structure, resulting in a poor separation of single strands of the two siRNA molecules using conventional d-IPRP-LC methods. Preliminary screening studies were first performed to identify the potentially critical method parameters to be included in the DoE study. A D-optimalDOCKET NO.: 38120-6046 (11853WO01) design DoE study including 26 runs was subsequently performed to systematically evaluate how different method parameters, including column temperature, mobile phase, gradient, and injection volume, affect the method performance, including peak resolution and peak shape. The critical analytical parameters impacting the separation resolution and peak shape were determined in this study, and, using optimal parameters determined in the DoE study, the baseline separation of siRNA single strands was achieved while maintaining appropriate peak shapes for purity characterization.

[0101] This DoE study provides a comprehensive understanding of how to improve performancefor single strand separation and informs future development of d-IPRP-LC methods for accurate and robust purity analysis for stability and release testing.

[0102] Unless described or defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, particular (yet not limiting) methods and materials are now described. General Definitions

[0103] 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.

[0104] As used herein, the term “chromatography” refers to a process in which a chemicalmixture comprising a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around, over, and / or through a stationary liquid or solid phase.

[0105] As used herein, the term “liquid chromatography” (LC) refers to a process in which abiological / 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 reversed phase (RP) liquid chromatography, ion-exchange (IEX) chromatography, size exclusion chromatography (SEC), affinity chromatography, hydrophobic interaction chromatography (HIC), hydrophilic interactionDOCKET NO.: 38120-6046 (11853WO01) chromatography (HILIC), or mixed-mode chromatography (MMC). In some embodiments, a sample can be subjected to any one of the aforementioned chromatographic methods or a combination thereof. Analytes separated using chromatography will feature distinctive retention times, reflecting the speed at which an analyte moves through the chromatographic column. Analytes may be compared using a chromatogram, which plots retention time on one axis and measured signal on another axis, where the measured signal may be produced from, for example, UV detection or fluorescence detection. In some exemplary embodiments, the methods and systems of the present disclosure include the use of denaturing ion pairing reverse phase liquid chromatography (d-IPRP-LC) ).

[0106] As used herein, the term “reverse phase liquid chromatography” (RP-LC) refers to aliquid chromatography that utilizes a nonpolar stationary phase and a polar mobile phase during binding of analyte to the stationary phase.

[0107] As used herein, the term “ion pairing reverse phase liquid chromatography” (IPRP-LC)refers to a variation of an RP-LC wherein an ion-pairing reagent (also sometimes called a counter-ion) is added to the mobile phase to better retain and separate highly polar or ionic compounds such as oligonucleotides on the stationary phase.

[0108] As used herein, the term “denaturing ion pairing reverse phase liquid chromatography”(d-IPRP-LC) refers to a variation of an IPRP-LC wherein the chromatography is run under denaturing conditions, such as high temperature, to separate double-stranded oligonucleotide molecules, such as siRNAs, into the two individual oligonucleotide strands.

[0109] As used herein, the term “retention time” refers to the length of time that a particularanalyte, such as an oligonucleotide, is retained by a liquid chromatography stationary phase prior to elution.

[0110] As used herein, the term “DoE” refers to the Design of Experiments that can befacilitated by selected instrument settings, multivariate analysis, and / or computer aided design and software. DoE allows for the screening of different combinations of parameters to find the ones that provide maximum response with respect to the desired output. Rather than evaluating “one-factor at a time” (OFAT), which is generally inefficient considering the number of possible experimental and instrument parameters, DoE allows for the simultaneous evaluation of an experiment’s or instrument’s many different parameters.DOCKET NO.: 38120-6046 (11853WO01)

[0111] As used herein, the term “D-optimal design” refers to a DoE set of parameters.Parameters may be deemed important based on previous user experience and previous literature. The process includes computer-assisted design, wherein a subset of all relevant combinations is chosen with a goal of maximizing D-efficiency of the design.

[0112] As used herein, the term “D-efficiency” refers to a computer aided design to decreaseworkload and provide a meaningful model or D-optimal design of experiment (DoE).

[0113] As used herein, the term “RNA” refers to ribonucleic acid. RNA molecules are essentialfor life and may be found in a variety of forms and functions. Long RNAs may include long non- coding RNA (lncRNA) and messenger RNA (mRNA), while small RNAs include transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), and small interfering RNA (siRNA), among many others. Synthetic RNA is useful for a variety of research and therapeutic functions, including for vaccines and gene therapy. An individual RNA polymer, or strand, may readily pair with a complementary RNA sequence. A first RNA strand and a second RNA strand with a complementary sequence to the first RNA strand may be defined as “sense” and “antisense”, respectively. In the case of an mRNA, the sense strand may be defined as the strand that is translated into an amino acid sequence. The complementary sequence would be considered the antisense strand.

[0114] As used herein, the term “oligonucleotide” refers to a short nucleic acid (DNA or RNA ora mixture of both) oligomer. Oligonucleotides may be found in nature or may be synthesized for a variety of applications. Oligonucleotides may have chemical modifications that are not typically found in nature. Nonlimiting examples of such chemical modifications are disclosed herein. Oligonucleotides may be single or double stranded. The term “oligomer” as used herein refers to is a molecule made up of a small number of repeating units, called monomers.

[0115] As used herein, the term “single-stranded oligonucleotide” refers to an oligonucleotidethat is not bound or hybridized to an oligonucleotide with a complementary sequence. Double- stranded oligonucleotides may be separated into single-stranded oligonucleotides by, for example, denaturing processes.

[0116] As used herein, the term “antisense oligonucleotide” (ASO) typically refers tooligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. AntisenseDOCKET NO.: 38120-6046 (11853WO01) oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. In certain embodiments, antisense oligonucleotides are single stranded.

[0117] As used herein, the term “small interfering RNA” (siRNA), also known as shortinterfering RNA, typically refers to a class of double-stranded RNA molecules, often about 20– 25 nucleotides in length, that plays a key role in the process of RNA interference (RNAi). siRNAs may include an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA, e.g., through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. As used herein, the term “short hairpin RNA” (shRNA) typically refers to an artificial RNA molecule comprising a hairpin that can be used to silence gene expression via RNA interference. Inhibitory Nucleic Acid Molecules

[0118] Inhibitory nucleic acid molecules are increasingly used as pharmaceutical drugcompounds to reduce or even eliminate expression of target genes in subjects. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules (also often referred to as antisense oligonucleotides or ASOs), small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). In principle, such inhibitory nucleic acid molecules can be designed to target any region of any target nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within a target genomic nucleic acid molecule or mRNA molecule and decreases expression of the target polypeptide in a cell in the subject. In some embodiments, the target inhibitor comprises an antisense molecule that hybridizes to a target genomic nucleic acid molecule or mRNA molecule and decreases expression of the target polypeptide in a cell in the subject. In some embodiments, the target inhibitor comprises an siRNA that hybridizes to a target genomic nucleic acid molecule or mRNA molecule and decreases expression of the target polypeptide in a cell in the subject. In some embodiments, the target inhibitor comprises an shRNA that hybridizes to a target genomic nucleic acid molecule or mRNA molecule and decreases expression of the target polypeptide in a cell in the subject.DOCKET NO.: 38120-6046 (11853WO01)

[0119] The inhibitory nucleic acid molecules can comprise RNA, DNA, or both RNA and DNA.The inhibitory nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence, such as in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecules can be within a vector or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. The inhibitory nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (such as, for example, fluorophore) or indirectly detectable (such as, for example, hapten, enzyme, or fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal. Oligonucleotide Modifications

[0120] In some embodiments, the inhibitory nucleic acid molecules comprise, for example,nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include a nucleotide that contains a modified base, sugar, or phosphate group, or that incorporates a non-natural moiety in its structure. Examples of non- natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, fluorophor-labeled nucleotides, glycol nucleic acid (GNA) nucleosides.

[0121] Nucleotide analogs can also include modifications of the base moiety. Modifications tothe base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo (such as, for example, 5-bromo), 5-trifluoromethyl andDOCKET NO.: 38120-6046 (11853WO01) other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0122] Nucleotide analogs can also include modifications of the sugar moiety. Modifications tothe sugar moiety include, but are not limited to, natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2’ position: H; OH; F; O-methyl; O-ethyl; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-10alkyl or C2-10alkenyl, and C2-10alkynyl. Exemplary 2’ sugar modifications also include, but are not limited to, -O[(CH2)nO]mCH3, -O(CH2)nOCH3, - O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)n-ONH2, and -O(CH2)nON[(CH2)nCH3)]2, where n and m, independently, are from 1 to about 10. Other modifications at the 2’ position include, but are not limited to, C1-10alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3’ position of the sugar on the 3’ terminal nucleotide or in 2’-5’ linked oligonucleotides and the 5’ position of 5’ terminal nucleotide. Modified sugars can also include those that contain modifications at the bridging ring oxygen, such as CH2and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0123] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphatemoieties include, but are not limited to, those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3’- alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3’- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate or modified phosphate linkage between two nucleotides can be through a 3’-5’ linkage or a 2’-5’ linkage, and the linkage can contain inverted polarity such as 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’.DOCKET NO.: 38120-6046 (11853WO01) Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0124] In some embodiments, the antisense nucleic acid molecules are gapmers, whereby thefirst one to seven nucleotides at the 5’ and 3’ ends each have 2’-methoxyethyl (2’-MOE) modifications. In some embodiments, the first five nucleotides at the 5’ and 3’ ends each have 2’-MOE modifications. In some embodiments, the first one to seven nucleotides at the 5’ and 3’ ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5’ and 3’ ends are RNA nucleotides. In some embodiments, each of the backbone linkages between the nucleotides is a phosphorothioate linkage.

[0125] In some embodiments, the siRNA molecules have termini modifications. In someembodiments, the 5’ end of the antisense strand is phosphorylated. In some embodiments, 5’- phosphate analogs that cannot be hydrolyzed, such as 5’-(E)-vinyl-phosphonate are used.

[0126] In some embodiments, the siRNA molecules have backbone modifications. In someembodiments, the modified phosphodiester groups that link consecutive ribose nucleosides have been shown to enhance the stability and in vivo bioavailability of siRNAs. The non-ester groups (-OH, =O) of the phosphodiester linkage can be replaced with sulfur, boron, or acetate to give phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, substituting the phosphodiester group with a phosphotriester can facilitate cellular uptake of siRNAs and retention on serum components by eliminating their negative charge.

[0127] In some embodiments, the siRNA molecules have sugar modifications. In someembodiments, the sugars are deprotonated (reaction catalyzed by exo- and endonucleases) whereby the 2’-hydroxyl can act as a nucleophile and attack the adjacent phosphorous in the phosphodiester bond. Such alternatives include 2’-O-methyl, 2’-O-methoxyethyl, and 2’-fluoro modifications. In some embodiments, one or more of the siRNA molecules has a glycol nucleic acid (GNA) modification. In some embodiments, one or more of the siRNA molecules has a 2’- deoxyribonucleoside modification.

[0128] In some embodiments, the siRNA molecules have base modifications. In someembodiments, the bases can be substituted with modified bases such as pseudouridine, 5’- methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine. In some embodiments, the siRNA molecules are modified with a glycol nucleic acid (GNA) nucleoside.DOCKET NO.: 38120-6046 (11853WO01)

[0129] In some embodiments, the siRNA molecules are conjugated to lipids. Lipids can beconjugated to the 5’ or 3’ termini of siRNA to improve their in vivo bioavailability by allowing them to associate with serum lipoproteins. Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids, such as palmitate and tocopherol. In some embodiments, one or more of the siRNA molecules comprise an oligonucleotide strand that is connected to a non-oligonucleotide moiety that comprises a GalNAc moiety. See, e.g., Friedrich & Aigner, BioDrugs, vol.36 (2022).

[0130] The term “GalNAc moiety” as used herein refers to an N-acetylgalactosamine orderivative thereof. Small Interfering RNA (siRNA) in Pharmaceutical Drug Development

[0131] RNA interference (RNAi) is a natural defense mechanism against exogenous genes frombacteria and viruses. RNAi modalities such as siRNA can knock down target gene expression in a sequence-specific manner by mediating targeted mRNA degradation. Since the establishment of the RNAi concept around 1998, a number of siRNA-based therapeutics are in development or have been approved for commercial use. The principal molecular and cellular mechanics of siRNA silencing of target genes are widely known and understood. See, e.g., Hu et al., Signal Transduction and Targeted Therapy, vol.5 (2020); Ebenezer et al., Int. J. Mol. Sci., vol.25 (2025); Guohua An, The Journal of Clinical Pharmacology, vol.64(1) (2024). Table 1 lists several siRNA Therapeutics that have been approved for commercial use or are in clinical development. Table 1: siRNA Therapeutics siRNA Therapeutics [Year Approved] siRNA Therapeutics in Clinical DevelopmentDOCKET NO.: 38120-6046 (11853WO01) Manufacture of siRNAs for Therapeutic Use

[0132] siRNAs can be manufactured in a variety of ways, including, but not limited to, chemicalsynthesis, in vitro transcription, digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, RNase III), expression in cells from an siRNA expression plasmid or viral vector, and expression in cells from a PCR-derived siRNA expression cassette.

[0133] Typically, siRNAs for human therapeutic use are chemically synthesized. Numerousapproaches for this synthesis are known in the art. See, e.g., US Patent No.7,872,118, which is incorporated herein by reference in its entirety. Denaturing Ion Pairing Reverse Phase Liquid Chromatography (d-IPRP-LC)

[0134] Therapeutic oligonucleotides have emerged as powerful tools in the treatment of complexdiseases, especially for conditions that lack efficacious treatment. Such therapeutic oligonucleotides include subclasses such as antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and short hairpin RNA (shRNA). Market-approval of such drugs by regulatory government agencies requires that these drugs satisfy stringent requirements with respect to their quality, identity, and integrity. As a result, a number of analytical methods have been developed for the characterization, such as liquid chromatography. Ion-pairing reversed-phase liquid chromatography (IPRP-LC) is a particular type of liquid chromatography wherein a hydrophobic stationary phase is combined with a mobile phase containing an ion-pair reagent. IPRP-LC can be operated under denaturing or non-denaturing conditions (d-IPRP-LC vs. nd-IPRP-LC). d- IPRP-LC is run at an elevated temperature to partially or completely denature double-stranded oligonucleotides. See, e.g., Fornstedt & Enmark, Journal of Chromatography Open, vol.3 (2023). d-IPRP-LC Stationary Phase

[0135] d-IPRP-LC typically comprises a stationary phase and a mobile phase. The concept anduse of a stationary phase for use in d-IPRP-LC is generally known in the art. The stationary phase is typically non-polar. Non-limiting examples of stationary phases are silica particles bonded with octadecyl (18-carbon) hydrocarbon chains, silica particles bonded with octyl (8- carbon) chains, silica particles bonded with phenyl groups or phenyl bonded via a hexyl linker, and silica particles bonded with cyano (-CN) groups. In an exemplary embodiment, the stationary phase is arranged in the form of a column. Stationary phases columns andDOCKET NO.: 38120-6046 (11853WO01) corresponding equipment and systems for d-IPRP-LC are commercially available from a variety of sources, including, but not limited to, Waters Corporation (Framingham, MA), Agilent Technologies, Inc. (Santa Clara, CA), ThermoFisher Scientific (Waltham, MA), and PerkinElmer, Inc. (Shelton, CT). Combinations of these exemplary stationary phases are also within the scope of the present disclosure. d-IPRP-LC Mobile Phase

[0136] d-IPRP-LC typically comprises a stationary phase and a mobile phase. The concept anduse of a mobile phase for use in d-IPRP-LC is generally known in the art. The elution order of analytes in the sample added to the stationary phase is governed by the polarity of the mobile phase. The mobile phase typically changes over time to facilitate the phase separation of different analytes in the sample. During loading of the sample and binding of the analytes to the stationary phase, the mobile phase is typically relatively polar. During the elution phase, the mobile phase is typically gradually made more non-polar. In an exemplary embodiment, a mobile phase in d-IPRP-LC consists primarily of a mixture of water or aqueous buffer, to which organic solvents are gradually added to elute analytes from a d-IPRP-LC stationary phase in a selective and sequential manner.

[0137] In an exemplary embodiment, the mobile phase for use in d-IPRP-LC is defined by a pairof two liquid components (mobile phase A and mobile phase B) that differ in their precise composition and in particular as to the degree of their polarity. The typically more polar mobile phase A is the mobile phase primarily used for the binding of the sample analyte to the stationary phase whereas increasing amounts of the less polar mobile phase B cause the elution of the sample analyte from the stationary phase in a gradual manner. The mobile phase is typically changed over the course of the d-IPRP-LC process to facilitate the gradual elution of the sample analytes based on their physical and chemical attributes. In an exemplary embodiment, this is achieved by blending the mobile phase A and the mobile phase B in a graded fashion to create a blended gradient of polarity within the mobile phase over time.

[0138] Non-limiting examples of components that may be used for the mobile phase include, butare not limited to, polar and / or aqueous solvents, non-polar and / or organic solvents, buffers, acids, ion-pairing reagents, and other additives. See, e.g., Boyes et al., “Modern Trends and Best Practices in Mobile-Phase Selection in Reversed-Phase Chromatography”, LCGC North America, vol.36 (10): 752–768 (2018).DOCKET NO.: 38120-6046 (11853WO01)

[0139] In some embodiments, mobile phase components include water, phosphate buffer,formate buffer, acetate buffer (e.g., ammonium acetate buffer), trifluoroacetic acid (TFA), methanol, acetonitrile, isopropanol, ethanol, tetrahydrofuran, tetrabutylammonium (TBA) salts, hexafluoroisopropanol (HFIP), triethylamine (TEA), ethylenediaminetetraacetic acid (EDTA), N,N-Diisopropylethylamine (DIEA), triethyl ammonium acetate, hexylammonium acetate, tetrabutyl ammonium acetate, tetrabutyl ammonium phosphate, or any combinations thereof. The aforesaid exemplary reagents are commercially available from a variety of sources commonly known.

[0140] Non-limiting examples of components that may be used for mobile phase A include, butare not limited to, primarily aqueous solvents, small amounts of organic solvents, buffers, acids, salts, ion-pairing reagents, other additives, or any combinations thereof.

[0141] Non-limiting examples of components that may be used for mobile phase B include, butare not limited to, primarily organic solvents, buffers, acids, salts, ion-pairing reagents, other additives, or any combinations thereof. d-IPRP-LC Ion-Pairing Reagents

[0142] The concept and use of an ion-pairing reagent for use in d-IPRP-LC is generally knownin the art. d-IPRP-LC is a type of RP-LC that can be used to separate hydrophilic or charged analytes on non-polar columns using reversed phase hydrophobic stationary phases. It can involve modifying the polarity of the charged analytes through their interaction with an ion- pairing reagent that is added to the mobile phase. In an exemplary embodiment, the ion-pair reagent is an alkylated amine. In an exemplary embodiment, the ion-pairing reagent includes triethylammonium acetate, hexyl ammonium acetate, tetrabutyl ammonium acetate, triethyl amine with hexafluoro isopropanol, or combinations thereof. The aforesaid exemplary reagents are commercially available from a variety of sources commonly known. d-IPRP-LC Mobile Phase Gradient

[0143] The concept and use of a mobile phase gradient for use in d-IPRP-LC is generally knownin the art. The mobile phase typically changes over time in a gradient-fashion to facilitate the phase separation of different analytes in the sample. During loading of the sample and binding of the analytes to the stationary phase, the mobile phase is typically relatively polar. During the elution phase, the mobile phase is typically gradually made more non-polar (e.g., through theDOCKET NO.: 38120-6046 (11853WO01) addition of methanol or any other non-polar solvent) to facilitate the gradual elution of the sample analytes based on their physical and chemical attributes. d-IPRP-LC Thermal Denaturation

[0144] The concept of thermal denaturation for use in d-IPRP-LC are generally known in the art.In some embodiments, thermal denaturation siRNA samples is achieved by the heating of stationary phase columns. Commercially available chromatography column systems that allow the columns to be heated, including columns for d-IPRP-LC, are readily available from a variety of vendors known in the art. Detection of Therapeutic Oligonucleotides by Ultra-Violet (UV) Light.

[0145] UV light is one of the most widely used detection principles for the analysis oftherapeutic oligonucleotides, such as siRNAs and their individual single oligonucleotide strands, by d-IPRP-LC. Typically, the term UV light refers to electromagnetic radiation of wavelengths of between 10 and 400 nanometers. Detection of analytes (e.g., oligonucleotides) that have been separated via liquid chromatography (e.g., d-IPRP-LC) utilizing ultraviolet (UV) light is commonly used in the art and corresponding equipment and technologies are readily available commercially. Such systems include, but are not limited single-wavelength detection systems, dual-wavelength detection systems, multi-wavelength detection systems, variable-wavelength detection systems, diode array detection (DAD) systems, and or photodiode array detection (PDA) detection systems. All of these systems might be suitable to practice the subject matter disclosed herein. Mass Spectrometry

[0146] The methods provided by this disclosure are suitable for use in conjunction with massspectrometry (MS). Therapeutic oligonucleotides of interest and one or more impurities such as truncated oligonucleotides, that have been separated via liquid chromatography may subsequently be analyzed by mass spectrometry (MS). Mass spectrometry (MS), and variants thereof, including tandem mass spectrometry (MS / MS), can be used to annotate and successful identify sequences and modifications of oligonucleotides used for therapeutic applications. See, e.g., US Patent No.12,099,042, which is incorporated herein by reference in its entirety. MS isDOCKET NO.: 38120-6046 (11853WO01) widely used and generally known in the field and appropriate equipment and protocols readily available commercially. Design of Experiment (DoE) Approach

[0147] Design of experiment (DoE) is a mathematical modeling approach extensively used forthe implementation of QbD in both research and industrial settings, including in pharmaceutical development. DoE is a structured, organized method for determining the relationships between factors affecting a process and the output of that process. DoE is the means of achieving process knowledge, through the establishment of mathematical relationships between process inputs and its outputs. See, e.g., Politis et al., Drug Development and Industrial Pharmacy, vol.43 (2017). Design of Experiment approaches as a general matter are known in the field. Dosage Forms

[0148] Therapeutic siRNAs are being developed or have been market-approved for use in avariety of dosage forms designed for, including, but not limited to, intravenous (IV) infusion, subcutaneous injection, intramuscular injection, inhalation, topical application, and intravitreal injection. See, e.g., Friedrich & Aigner, BioDrugs, vol.36 (2022). siRNA therapeutics are often delivered via lipid nano particles. In some embodiments, siRNA dosage forms are fixed dose combinations of at least two small interfering RNAs (siRNAs). Appropriate dosage forms and delivery vehicles for siRNA therapeutic products are known in the field. Exemplary Embodiments

[0149] This disclosure provides methods for separating at least two oligonucleotides in a sample.

[0150] The terms “separating” or “separate” or the like as used herein refer to the process ofdifferentially moving individual components of a mixture through a stationary phase (i.e., a column) at different speeds, so that they emerge from the stationary phase at different times (different retention times).

[0151] The terms “oligonucleotides” or “oligonucleotide” refer to a short nucleic acid (DNA orRNA or a mixture of both) oligomer(s). Oligonucleotides may be found in nature or may be synthesized for a variety of applications. Oligonucleotides may have modifications that are not typically found in nature. Nonlimiting examples of such modifications are disclosed herein. Oligonucleotides may be single or double stranded. In preferred embodiments, theDOCKET NO.: 38120-6046 (11853WO01) oligonucleotide is a sense or antisense strand in an siRNA molecule. In more preferred embodiments, the oligonucleotide is a sense or antisense strand in an siRNA molecule, wherein the oligonucleotide has non-natural modifications.

[0152] In some exemplary embodiments, the methods can comprise obtaining a samplecomprising at least two oligonucleotides, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotides, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

[0153] The term “sample” as used herein has the ordinary meaning generally understood in theart. The term may include a solution or mixture containing the analytes of interest that is introduced into a chromatography system for separation, identification, and / or quantification.

[0154] In some embodiments, the column temperature is from about 75 °C to about 85 °C, fromabout 75 °C to about 90 °C, from about 80 °C to about 90 °C, from about 75 °C to about 77.5 °C, from about 77.5 °C to about 80 °C, from about 80 °C to about 82.5 °C, from about 82.5 °C to about 85 °C, from about 85 °C to about 87.5 °C, from about 87.5 °C to about 90 °C, from about 90 °C to about 92.5 °C, or from about 92.5 °C to about 95 °C.

[0155] In some embodiments, the column temperature is from about 75 °C to about 85 °C, fromabout 75 °C to about 90 °C, from about 75 °C to about 95 °C, from about 77.5 °C to about 92.5 °C, from about 80 °C to about 90 °C, from about 82.5 °C to about 87.5 °C, from about 83 °C to about 87 °C, or from about 84 °C to about 86 °C.

[0156] In some embodiments, the column temperature is from 75 °C to 85 °C, from 75 °C to 90°C, from 80 °C to 90 °C, from 75 °C to 77.5 °C, from 77.5 °C to 80 °C, from 80 °C to 82.5 °C, from 82.5 °C to 85 °C, from 85 °C to 87.5 °C, from 87.5 °C to 90 °C, from 90 °C to 92.5 °C, or from 92.5 °C to 95 °C.

[0157] In some embodiments, the column temperature is from 75 °C to 85 °C, from 75 °C to 90°C, from 75 °C to 95 °C, from 77.5 °C to 92.5 °C, from 80 °C to 90 °C, from 82.5 °C to 87.5 °C, from 83 °C to 87 °C, or from 84 °C to 86 °C.DOCKET NO.: 38120-6046 (11853WO01)

[0158] In some embodiments, the injection volume is from about 2 µL to about 8 µL, from about1 µL to about 8 µL, from about 1 µL to about 3 µL, from about 0.1 µL to about 0.2 µL, from about 0.2 µL to about 0.3 µL, from about 0.3 µL to about 0.4 µL, from about 0.4 µL to about 0.5 µL, from about 0.5 µL to about 0.6 µL, from about 0.6 µL to about 0.7 µL, from about 0.7 µL to about 0.8 µL, from about 0.8 µL to about 0.9 µL, from about 0.9 µL to about 1 µL, from about 1 µL to about 1.1 µL, from about 1.1 µL to about 1.2 µL, from about 1.2 µL to about 1.3 µL, from about 1.3 µL to about 1.4 µL, from about 1.4 µL to about 1.5 µL, from about 1.5 µL to about 1.6 µL, from about 1.6 µL to about 1.7 µL, from about 1.7 µL to about 1.8 µL, from about 1.8 µL to about 1.9 µL, from about 1.9 µL to about 2 µL, from about 2 µL to about 2.1 µL, from about 2.1 µL to about 2.2 µL, from about 2.2 µL to about 2.3 µL, from about 2.3 µL to about 2.4 µL, from about 2.4 µL to about 2.5 µL, from about 2.5 µL to about 2.6 µL, from about 2.6 µL to about 2.7 µL, from about 2.7 µL to about 2.8 µL, from about 2.8 µL to about 2.9 µL, from about 2.9 µL to about 3 µL, from about 3 µL to about 3.1 µL, from about 3.1 µL to about 3.2 µL, from about 3.2 µL to about 3.3 µL, from about 3.3 µL to about 3.4 µL, from about 3.4 µL to about 3.5 µL, from about 3.5 µL to about 3.6 µL, from about 3.6 µL to about 3.7 µL, from about 3.7 µL to about 3.8 µL, or from about 3.8 µL to about 3.9 µL.

[0159] In some embodiments, the injection volume is from about 2 µL to about 8 µL, from about1 µL to about 8 µL, from about 0.1 µL to about 3.9 µL, from about 0.2 µL to about 3.8 µL, from about 0.3 µL to about 3.7 µL, from about 0.4 µL to about 3.6 µL, from about 0.5 µL to about 3.5 µL, from about 0.6 µL to about 3.4 µL, from about 0.7 µL to about 3.3 µL, from about 0.8 µL to about 3.2 µL, from about 0.9 µL to about 3.1 µL, from about 1 µL to about 3 µL, from about 1.1 µL to about 2.9 µL, from about 1.2 µL to about 2.8 µL, from about 1.3 µL to about 2.7 µL, from about 1.4 µL to about 2.6 µL, from about 1.5 µL to about 2.5 µL, from about 1.6 µL to about 2.4 µL, from about 1.7 µL to about 2.3 µL, from about 1.8 µL to about 2.2 µL, or from about 1.9 µL to about 2.1 µL.

[0160] In some embodiments, the injection volume is from 2 µL to 8 µL, from 1 µL to 8 µL,from 1 µL to 3 µL, from 0.1 µL to 0.2 µL, from 0.2 µL to 0.3 µL, from 0.3 µL to 0.4 µL, from 0.4 µL to 0.5 µL, from 0.5 µL to 0.6 µL, from 0.6 µL to 0.7 µL, from 0.7 µL to 0.8 µL, from 0.8 µL to 0.9 µL, from 0.9 µL to 1 µL, from 1 µL to 1.1 µL, from 1.1 µL to 1.2 µL, from 1.2 µL to 1.3 µL, from 1.3 µL to 1.4 µL, from 1.4 µL to 1.5 µL, from 1.5 µL to 1.6 µL, from 1.6 µL to 1.7 µL, from 1.7 µL to 1.8 µL, from 1.8 µL to 1.9 µL, from 1.9 µL to 2 µL, from 2 µL to 2.1 µL,DOCKET NO.: 38120-6046 (11853WO01) from 2.1 µL to 2.2 µL, from 2.2 µL to 2.3 µL, from 2.3 µL to 2.4 µL, from 2.4 µL to 2.5 µL, from 2.5 µL to 2.6 µL, from 2.6 µL to 2.7 µL, from 2.7 µL to 2.8 µL, from 2.8 µL to 2.9 µL, from 2.9 µL to 3 µL, from 3 µL to 3.1 µL, from 3.1 µL to 3.2 µL, from 3.2 µL to 3.3 µL, from 3.3 µL to 3.4 µL, from 3.4 µL to 3.5 µL, from 3.5 µL to 3.6 µL, from 3.6 µL to 3.7 µL, from 3.7 µL to 3.8 µL, or from 3.8 µL to 3.9 µL.

[0161] In some embodiments, the injection volume is from 2 µL to 8 µL, from 1 µL to 8 µL,from 0.1 µL to 3.9 µL, from 0.2 µL to 3.8 µL, from 0.3 µL to 3.7 µL, from 0.4 µL to 3.6 µL, from 0.5 µL to 3.5 µL, from 0.6 µL to 3.4 µL, from 0.7 µL to 3.3 µL, from 0.8 µL to 3.2 µL, from 0.9 µL to 3.1 µL, from 1 µL to 3 µL, from 1.1 µL to 2.9 µL, from 1.2 µL to 2.8 µL, from 1.3 µL to 2.7 µL, from 1.4 µL to 2.6 µL, from 1.5 µL to 2.5 µL, from 1.6 µL to 2.4 µL, from 1.7 µL to 2.3 µL, from 1.8 µL to 2.2 µL, or from 1.9 µL to 2.1 µL.

[0162] In preferred embodiments, the mobile phase B contains EDTA at a concentration of 5µM in methanol. In even more preferred embodiments, the mobile phase B contains only methanol that contains EDTA at a concentration of 5 µM.

[0163] In some embodiments, the mobile phase B comprises ethylenediaminetetraacetic acid(EDTA) in methanol and one or more other components that include, but are not limited to, water, phosphate buffer, formate buffer, acetate buffer (e.g., ammonium acetate buffer), trifluoroacetic acid (TFA), methanol, acetonitrile, isopropanol, ethanol, tetrahydrofuran, tetrabutylammonium (TBA) salts, hexafluoroisopropanol (HFIP), triethylamine (TEA), ethylenediaminetetraacetic acid (EDTA), N,N-Diisopropylethylamine (DIEA), triethyl ammonium acetate, hexylammonium acetate, tetrabutyl ammonium acetate, tetrabutyl ammonium phosphate, or any combinations thereof, or any other component commonly used in the mobile phase B of d-IPRP-LC.

[0164] In some embodiments, the mobile phase B comprises ethylenediaminetetraacetic acid(EDTA) at a concentration of about 5 μM. In some embodiments, the mobile phase B comprises ethylenediaminetetraacetic acid (EDTA) at a concentration of from about 3 μM to about 7 μM, from about 3 μM to about 3.2 μM, from about 3.2 μM to about 3.4 μM, from about 3.4 μM to about 3.6 μM, from about 3.6 μM to about 3.8 μM, from about 3.8 μM to about 4 μM, from about 4 μM to about 4.2 μM, from about 4.2 μM to about 4.4 μM, from about 4.4 μM to about 4.6 μM, from about 4.6 μM to about 4.8 μM, from about 4.8 μM to about 5 μM, from about 5 μM to about 5.2 μM, from about 5.2 μM to about 5.4 μM, from about 5.4 μM to about 5.6 μM,DOCKET NO.: 38120-6046 (11853WO01) from about 5.6 μM to about 5.8 μM, from about 5.8 μM to about 6 μM, from about 6 μM to about 6.2 μM, from about 6.2 μM to about 6.4 μM, from about 6.4 μM to about 6.6 μM, from about 6.6 μM to about 6.8 μM, or from about 6.8 μM to about 7 μM.

[0165] In some embodiments, the mobile phase B comprises ethylenediaminetetraacetic acid(EDTA) at a concentration of from about 3 μM to about 7 μM, from about 3.1 μM to about 6.9 μM, from about 3.2 μM to about 6.8 μM, from about 3.3 μM to about 6.7 μM, from about 3.4 μM to about 6.6 μM, from about 3.5 μM to about 6.5 μM, from about 3.6 μM to about 6.4 μM, from about 3.7 μM to about 6.3 μM, from about 3.8 μM to about 6.2 μM, from about 3.9 μM to about 6.1 μM, from about 4 μM to about 6 μM, from about 4.1 μM to about 5.9 μM, from about 4.2 μM to about 5.8 μM, from about 4.3 μM to about 5.7 μM, from about 4.4 μM to about 5.6 μM, from about 4.5 μM to about 5.5 μM, from about 4.6 μM to about 5.4 μM, from about 4.7 μM to about 5.3 μM, from about 4.8 μM to about 5.2 μM, or from about 4.9 μM to about 5.1 μM.

[0166] In some embodiments, the mobile phase B comprises ethylenediaminetetraacetic acid(EDTA) at a concentration of 5 μM. In some embodiments, the mobile phase B comprises ethylenediaminetetraacetic acid (EDTA) at a concentration of from 3 μM to 7 μM, from 3 μM to 3.2 μM, from 3.2 μM to 3.4 μM, from 3.4 μM to 3.6 μM, from 3.6 μM to 3.8 μM, from 3.8 μM to 4 μM, from 4 μM to 4.2 μM, from 4.2 μM to 4.4 μM, from 4.4 μM to 4.6 μM, from 4.6 μM to 4.8 μM, from 4.8 μM to 5 μM, from 5 μM to 5.2 μM, from 5.2 μM to 5.4 μM, from 5.4 μM to 5.6 μM, from 5.6 μM to 5.8 μM, from 5.8 μM to 6 μM, from 6 μM to 6.2 μM, from 6.2 μM to 6.4 μM, from 6.4 μM to 6.6 μM, from 6.6 μM to 6.8 μM, or from 6.8 μM to 7 μM.

[0167] In some embodiments, the mobile phase B comprises ethylenediaminetetraacetic acid(EDTA) at a concentration of from 3 μM to 7 μM, from 3.1 μM to 6.9 μM, from 3.2 μM to 6.8 μM, from 3.3 μM to 6.7 μM, from 3.4 μM to 6.6 μM, from 3.5 μM to 6.5 μM, from 3.6 μM to 6.4 μM, from 3.7 μM to 6.3 μM, from 3.8 μM to 6.2 μM, from 3.9 μM to 6.1 μM, from 4 μM to 6 μM, from 4.1 μM to 5.9 μM, from 4.2 μM to 5.8 μM, from 4.3 μM to 5.7 μM, from 4.4 μM to 5.6 μM, from 4.5 μM to 5.5 μM, from 4.6 μM to 5.4 μM, from 4.7 μM to 5.3 μM, from 4.8 μM to 5.2 μM, or from 4.9 μM to 5.1 μM.

[0168] In some embodiments, the initial concentration of the mobile phase B is from about 3%to about 7%. In some embodiments, the initial concentration of the mobile phase B is from about 5% to about 10%. In some embodiments, the initial concentration of the mobile phase B is from about 3% to about 10%. In some embodiments, the initial concentration of the mobile phase B isDOCKET NO.: 38120-6046 (11853WO01) from about 3% to about 3.2%, from about 3.2% to about 3.4%, from about 3.4% to about 3.6%, from about 3.6% to about 3.8%, from about 3.8% to about 4%, from about 4% to about 4.2%, from about 4.2% to about 4.4%, from about 4.4% to about 4.6%, from about 4.6% to about 4.8%, from about 4.8% to about 5%, from about 5% to about 5.2%, from about 5.2% to about 5.4%, from about 5.4% to about 5.6%, from about 5.6% to about 5.8%, from about 5.8% to about 6%, from about 6% to about 6.2%, from about 6.2% to about 6.4%, from about 6.4% to about 6.6%, from about 6.6% to about 6.8%, or from about 6.8% to about 7%.

[0169] In some embodiments, the initial concentration of the mobile phase B is from about 3.1%to about 6.9%, from about 3.2% to about 6.8%, from about 3.3% to about 6.7%, from about 3.4% to about 6.6%, from about 3.5% to about 6.5%, from about 3.6% to about 6.4%, from about 3.7% to about 6.3%, from about 3.8% to about 6.2%, from about 3.9% to about 6.1%, from about 4% to about 6%, from about 4.1% to about 5.9%, from about 4.2% to about 5.8%, from about 4.3% to about 5.7%, from about 4.4% to about 5.6%, from about 4.5% to about 5.5%, from about 4.6% to about 5.4%, from about 4.7% to about 5.3%, from about 4.8% to about 5.2%, or from about 4.9% to about 5.1%.

[0170] In some embodiments, the initial concentration of the mobile phase B is from 3% to 7%.In some embodiments, the initial concentration of the mobile phase B is from 5% to 10%. In some embodiments, the initial concentration of the mobile phase B is from 3% to 10%. In some embodiments, the initial concentration of the mobile phase B is from 3% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from 3.8% to 4%, from 4% to 4.2%, from 4.2% to 4.4%, from 4.4% to 4.6%, from 4.6% to 4.8%, from 4.8% to 5%, from 5% to 5.2%, from 5.2% to 5.4%, from 5.4% to 5.6%, from 5.6% to 5.8%, from 5.8% to 6%, from 6% to 6.2%, from 6.2% to 6.4%, from 6.4% to 6.6%, from 6.6% to 6.8%, or from 6.8% to 7%.

[0171] In some embodiments, the initial concentration of the mobile phase B is from 3.1% to6.9%, from 3.2% to 6.8%, from 3.3% to 6.7%, from 3.4% to 6.6%, from 3.5% to 6.5%, from 3.6% to 6.4%, from 3.7% to 6.3%, from 3.8% to 6.2%, from 3.9% to 6.1%, from 4% to 6%, from 4.1% to 5.9%, from 4.2% to 5.8%, from 4.3% to 5.7%, from 4.4% to 5.6%, from 4.5% to 5.5%, from 4.6% to 5.4%, from 4.7% to 5.3%, from 4.8% to 5.2%, or from 4.9% to 5.1%.

[0172] In some embodiments, the maximum concentration of the mobile phase B is from about18% to about 26%. In some embodiments, the maximum concentration of the mobile phase B is from about 22% to about 27%. In some embodiments, the maximum concentration of the mobileDOCKET NO.: 38120-6046 (11853WO01) phase B is from about 18% to about 27%. In some embodiments, the maximum concentration of the mobile phase B is from about 18% to about 18.5%, from about 18.5% to about 19%, from about 19% to about 19.5%, from about 19.5% to about 20%, from about 20% to about 20.5%, from about 20.5% to about 21%, from about 21% to about 21.5%, from about 21.5% to about 21.75%, from about 21.75% to about 22.25%, from about 22.25% to about 22.5%, from about 22.5% to about 23%, from about 23% to about 23.5%, from about 23.5% to about 24%, from about 24% to about 24.5%, from about 24.5% to about 25%, from about 25% to about 25.5%, or from about 25.5% to about 26%.

[0173] In some embodiments, the maximum concentration of the mobile phase B is from about18% to about 26%, from about 18.5% to about 25.5%, from about 19% to about 25%, from about 19.5% to about 24.5%, from about 20% to about 24%, from about 20.5% to about 23.5%, from about 21% to about 23%, from about 21.5% to about 22.5%, from about 21.6% to about 22.4%, from about 21.7% to about 22.3%, from about 21.8% to about 22.2%, or from about 21.9% to about 22.1%.

[0174] In some embodiments, the maximum concentration of the mobile phase B is from 18% to26%. In some embodiments, the maximum concentration of the mobile phase B is from 22% to 27%. In some embodiments, the maximum concentration of the mobile phase B is from 18% to 27%. In some embodiments, the maximum concentration of the mobile phase B is from 18% to 18.5%, from 18.5% to 19%, from 19% to 19.5%, from 19.5% to 20%, from 20% to 20.5%, from 20.5% to 21%, from 21% to 21.5%, from 21.5% to 21.75%, from 21.75% to 22.25%, from 22.25% to 22.5%, from 22.5% to 23%, from 23% to 23.5%, from 23.5% to 24%, from 24% to 24.5%, from 24.5% to 25%, from 25% to 25.5%, or from 25.5% to 26%.

[0175] In some embodiments, the maximum concentration of the mobile phase B is from 18% to26%, from 18.5% to 25.5%, from 19% to 25%, from 19.5% to 24.5%, from 20% to 24%, from 20.5% to 23.5%, from 21% to 23%, from 21.5% to 22.5%, from 21.6% to 22.4%, from 21.7% to 22.3%, from 21.8% to 22.2%, or from 21.9% to 22.1%.

[0176] In some embodiments, the initial concentration of the mobile phase B is from about 3.5%to about 6.5% and the maximum concentration of the mobile phase B is from about 19% to about 25%, the initial concentration of the mobile phase B is from about 4% to about 6% and the maximum concentration of the mobile phase B is from about 20% to about 24%, the initial concentration of the mobile phase B is from about 4.5% to about 5.5% and the maximumDOCKET NO.: 38120-6046 (11853WO01) concentration of the mobile phase B is from about 19% to about 25%, the initial concentration of the mobile phase B is from about 4.75% to about 5.25% and the maximum concentration of the mobile phase B is from about 20% to about 24%, the initial concentration of the mobile phase B is from about 4.75% to about 5.25% and the maximum concentration of the mobile phase B is from about 21% to about 23%, the initial concentration of the mobile phase B is from about 4.75% to about 5.25% and the maximum concentration of the mobile phase B is from about 21.5% to about 22.5%, the initial concentration of the mobile phase B is from about 4.75% to about 5.25% and the maximum concentration of the mobile phase B is from about 21.75% to about 22.25%, or the initial concentration of the mobile phase B is from about 4.9% to about 5.1% and the maximum concentration of the mobile phase B is from about 21.9% to about 22.1%.

[0177] In some embodiments, the initial concentration of the mobile phase B is from 3.5% to6.5% and the maximum concentration of the mobile phase B is from 19% to 25%, the initial concentration of the mobile phase B is from 4% to 6% and the maximum concentration of the mobile phase B is from 20% to 24%, the initial concentration of the mobile phase B is from 4.5% to 5.5% and the maximum concentration of the mobile phase B is from 19% to 25%, the initial concentration of the mobile phase B is from 4.75% to 5.25% and the maximum concentration of the mobile phase B is from 20% to 24%, the initial concentration of the mobile phase B is from 4.75% to 5.25% and the maximum concentration of the mobile phase B is from 21% to 23%, the initial concentration of the mobile phase B is from 4.75% to 5.25% and the maximum concentration of the mobile phase B is from 21.5% to 22.5%, the initial concentration of the mobile phase B is from 4.75% to 5.25% and the maximum concentration of the mobile phase B is from 21.75% to 22.25%, or the initial concentration of the mobile phase B is from 4.9% to 5.1% and the maximum concentration of the mobile phase B is from 21.9% to 22.1%.

[0178] The term “concentration of the mobile phase B” as used herein refers to the percentproportion of mobile phase B used to create the mobile phase made by blending a volume of mobile phase A and mobile phase B.

[0179] The term “initial concentration” as used herein refers to the time point T=0min when themobile phase B is first blended into the flow of mobile phase A to initiate the elution gradient during a chromatography run. The term “maximum concentration” as used herein refers to the concentration at the end of the separation phase. In some embodiments, the term “maximumDOCKET NO.: 38120-6046 (11853WO01) concentration” as used herein therefore refers to the time point T=40min (40 minutes post T=0min) when the maximum amount of mobile phase B is blended with mobile phase A at the end of the elution gradient / separation gradient during a chromatography run, just before the chromatography column is flushed with a very high concentration of mobile phase B.

[0180] In some embodiments, said at least two oligonucleotides are sense strands of smallinterfering RNAs. In some embodiments, said at least two oligonucleotides are antisense strands of small interfering RNAs. The term “antisense strand” (also abbreviated “AS” herein)(also referred to as “guide strand”) with respect to an siRNA molecule refers to an oligonucleotide that has a nucleotide sequence that is complementary to the sequence of an RNA molecule carrying translatable genetic code. The term “sense strand” (also abbreviated “SS” herein)(also referred to as “passenger strand”) with respect to an siRNA molecule refers to an oligonucleotide that is complementary to the antisense strand.

[0181] The sense strand and the antisense strand may have different length. Either one or both ofthe sense strand and the antisense strand may have one or more non-natural modifications. Either one or both of the sense strand and the antisense strand may be connected to a non- oligonucleotide moiety.

[0182] In some embodiments, the sample is a fixed dose combination of at least two smallinterfering RNAs (siRNAs). The term “fixed dose combination” as used herein refers to a single dosage form (e.g., a tablet, capsule, injection, or other) containing a particular combination of two or more active pharmaceutical ingredients (APIs) in fixed proportions. The fixed dose combination of these embodiments may be any type of dosage form that is suitable for the administration of siRNAs. Such dosage forms include, but are not limited to, dosage forms for parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular administration.

[0183] In some embodiments, said at least two oligonucleotides are detected using ultraviolet(UV) detection. Typically, the term UV light refers to electromagnetic radiation of wavelengths of between 10 and 400 nanometers. Detection of analytes (e.g., oligonucleotides) that have been separated via liquid chromatography (e.g., d-IPRP-LC) utilizing ultraviolet (UV) light is commonly used in the art and corresponding equipment and technologies are readily available commercially. Any suitable UV-detection system falls within the scope of the instant disclosure. In a preferred embodiment, the wavelength for nucleic acid, e.g., RNA, detection is 260 nm.DOCKET NO.: 38120-6046 (11853WO01)

[0184] Such systems include, but are not limited single-wavelength detection systems, dual-wavelength detection systems, multi-wavelength detection systems, variable-wavelength detection systems, diode array detection (DAD) systems, and or photodiode array detection (PDA) detection systems. Such systems further include, but are not limited to, the an ACQUITY UPLC H-Class PLUS System equipped with a tunable ultraviolet (TUV) detector and Empower®3 Software by Waters Corporation (Milford, MA).

[0185] In some embodiments, conditions for the separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water. In some embodiments, hexafluoro isopropanol is present in the mobile phase A at a concentration of 550 mM, triethylamine is present in the mobile phase A at a concentration of 13 mM, and EDTA is present in the mobile phase A at a concentration of 5 µM. In some embodiments, hexafluoro isopropanol, triethylamine, and EDTA are dissolved in a 9:1 mixture of H2O and methanol. In preferred embodiments, the mobile phase A is a 9:1 mixture of H2O and methanol that contains hexafluoro isopropanol at a concentration of 550 mM, triethylamine at a concentration of 13 mM, and EDTA at a concentration of 5 µM. In even more preferred embodiments, the mobile phase A contains only a 9:1 mixture of H2O and methanol that contains hexafluoro isopropanol at a concentration of 550 mM, triethylamine at a concentration of 13 mM, and EDTA at a concentration of 5 µM. In some embodiments, the mobile phase A includes hexafluoro isopropanol, triethylamine, and EDTA in a 9:1 mixture of H2O:methanol, as well as one or more other components that include, but are not limited to, phosphate buffer, formate buffer, acetate buffer (e.g., ammonium acetate buffer), trifluoroacetic acid (TFA), methanol, acetonitrile, isopropanol, ethanol, tetrahydrofuran, tetrabutylammonium (TBA) salts, N,N- Diisopropylethylamine (DIEA), triethyl ammonium acetate, hexylammonium acetate, tetrabutyl ammonium acetate, tetrabutyl ammonium phosphate, or any combinations thereof, or any other component commonly used in the mobile phase A of d-IPRP-LC.

[0186] In some embodiments, a total concentration of oligonucleotides in the sample is about 0.4mg / mL. In some embodiments, a total concentration of oligonucleotides in the sample is about 0.4 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL. In some embodiments, a total concentration of oligonucleotides in the sample is 0.4 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL,DOCKET NO.: 38120-6046 (11853WO01) 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL. In a preferred embodiment, total concentration of oligonucleotides is determined using UV detection at 260 nm.

[0187] In some embodiments, a column for the denaturing ion pairing liquid chromatographyseparation is a C18 column. The term “C18 column” as used herein refers to a stationary phase wherein made of silica particles chemically bonded to octadecyl (C18) hydrocarbon chains. C18 chromatography columns are known in the art and readily available from various commercial vendors, such as for example Waters Corporation (Milford, MA). All of these different commercially available C18 chromatography columns are generally suitable for the methods disclosed herein. In preferred embodiments, the C18 column is the Acquity Premier Oligonucleotides BEH C18 (300 Å, 1.7 µm, 2.1 mm x 150 mm) column by Waters Corporation (Milford, MA).

[0188] In some embodiments, the column temperature is about 85 °C. Chromatography columnsare heated to perform the chromatography, e.g., d-IPRP-LC, under denaturing conditions. Such conditions may, for example, facilitate the separation of the oligonucleotide double-strands of siRNA molecules during the chromatographic process, and allow the detection of individual oligonucleotide strands as opposed to double-stranded siRNA molecules. Commercially available chromatography column systems that allow the columns to be heated, including columns for d-IPRP-LC, are readily available from a variety of vendors known in the art.

[0189] In some embodiments, the injection volume is about 2 µL. The term “injection volume”as used herein refers to the volume of a sample injected into a stationary phase, e.g., a chromatography column, before the run of the chromatography process.

[0190] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0191] This disclosure additionally provides methods for separating at least two oligonucleotidestrands in a sample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising at least two oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initialDOCKET NO.: 38120-6046 (11853WO01) concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

[0192] The term “oligonucleotide strand” refers to a chain of nucleotides connected by covalentbonds. An oligonucleotide strand can be DNA, RNA, or both, and can also include modifications that are not typically found in nature.

[0193] In some embodiments, said at least two oligonucleotide strands are sense strands of smallinterfering RNAs. In some embodiments, said at least two oligonucleotide strands are antisense strands of small interfering RNAs.

[0194] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0195] In some embodiments, said at least two oligonucleotide strands are detected usingultraviolet detection.

[0196] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.

[0197] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0198] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.

[0199] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.

[0200] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0201] This disclosure further provides methods for separating a first single-strandedoligonucleotide from at least one additional single-stranded oligonucleotide. In some exemplary embodiments, the methods can comprise obtaining a sample comprising a first single-stranded oligonucleotide and at least one additional single-stranded oligonucleotide, and subjecting said sample to denaturing ion pairing liquid chromatography separation, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B isDOCKET NO.: 38120-6046 (11853WO01) from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

[0202] As used herein, the term “single-stranded oligonucleotide” refers to an oligonucleotidethat is not bound or hybridized to a complementary sequence.

[0203] In some embodiments, said first single-stranded oligonucleotide and said at least oneadditional single-stranded oligonucleotide are sense strands of small interfering RNAs. In some embodiments, said first single-stranded oligonucleotide and said at least one additional single- stranded oligonucleotide are antisense strands of small interfering RNAs.

[0204] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0205] In some embodiments, said single-stranded oligonucleotides are detected using ultravioletdetection.

[0206] In some embodiments, conditions for said separation further include a mobile phase Acomprising hexafluoro isopropanol, triethylamine, and EDTA in water.

[0207] In some embodiments, a total concentration of oligonucleotides in said sample is about0.4 mg / mL.

[0208] In some embodiments, a column for said denaturing ion pairing liquid chromatographyseparation is a C18 column.

[0209] In some embodiments, the column temperature is about 85 °C. In some embodiments, theinjection volume is about 2 µL.

[0210] In some embodiments, an initial concentration of the mobile phase B is about 5%. Insome embodiments, a maximum concentration of the mobile phase B is about 22%. In a further embodiment, a maximum concentration of the mobile phase B is at about 40 minutes.

[0211] This disclosure provides methods for separating at least two oligonucleotide strands in asample. In some exemplary embodiments, the methods can comprise obtaining a sample comprising at least two oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of saidDOCKET NO.: 38120-6046 (11853WO01) mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%.

[0212] In some embodiments, said at least two oligonucleotide strands are antisense strands ofsmall interfering RNAs.

[0213] In some embodiments, said sample is a fixed dose combination of at least two smallinterfering RNAs.

[0214] This disclosure provides methods for separating four oligonucleotide strands in a sample.In some exemplary embodiments, the methods can comprise obtaining a sample comprising four oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%, wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA.

[0215] In some embodiments, each of said two small interfering RNAs comprises anoligonucleotide strand that is connected to a non-oligonucleotide moiety. In some embodiments, said non-oligonucleotide moiety comprises a GalNAc moiety.

[0216] The term “GalNAc moiety” as used herein refers to an N-Acetylgalactosamine orderivative thereof. Non-oligonucleotide moieties may comprise more than one GalNAc moiety. In some exemplary embodiments, the non-oligonucleotide moiety comprises one GalNAc moiety. In some exemplary embodiments, the non-oligonucleotide moiety comprises one GalNAc moiety, two GalNAc moieties, three GalNAc moieties, four GalNAc moieties, five GalNAc moieties, six GalNAc moieties, seven GalNAc moieties, eight GalNAc moieties, nine GalNAc moieties, or ten GalNAc moieties (monovalent, bivalent, trivalent, tetravalent etc.).

[0217] In some embodiments, each of said four oligonucleotide strands comprises aphosphorothioate (PS) backbone modification.

[0218] The term “backbone” as used herein in connection with oligonucleotides refers to thealternating sugar and phosphate groups of a naturally occurring oligonucleotide. The term “a phosphorothioate (PS) backbone modification” as used herein refers to the backbone of anDOCKET NO.: 38120-6046 (11853WO01) oligonucleotide wherein an oxygen atom of one or more phosphate groups has is replaced with a sulfur atom.

[0219] This disclosure provides methods for separating four oligonucleotide strands in a sample.In some exemplary embodiments, the methods can comprise obtaining a sample comprising four oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%, wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA, wherein each small interfering RNA comprises an oligonucleotide strand that is connected to a non-oligonucleotide moiety that comprises a GalNAc moiety, and wherein each of said four oligonucleotide strands comprises a phosphorothioate (PS) backbone modification.

[0220] This disclosure provides methods for separating four oligonucleotide strands in a sample.In some exemplary embodiments, the methods can comprise obtaining a sample comprising four oligonucleotide strands, and subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of 85 °C, an injection volume of 2 µL, a mobile phase B comprising 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at T=0min an initial concentration of said mobile phase B is 5% and at T=40min a concentration of said mobile phase B is 22%, wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA, wherein each of said two small interfering RNAs comprises an oligonucleotide strand that is connected to a non-oligonucleotide moiety that comprises a GalNAc moiety, and wherein each of said four oligonucleotide strands comprises a phosphorothioate (PS) backbone modification.

[0221] All patents, patent applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.DOCKET NO.: 38120-6046 (11853WO01)

[0222] The present disclosure will be more fully understood by reference to the followingexamples. They should not, however, be construed as limiting the scope of the disclosure. REFERENCES

[0223] Blevins et al., Revealing New Analytical Insights Into RNA Complexes: Divalent siRNACharacterization by Liquid Chromatography and Mass Spectrometry, Analytical Chemistry, Vol. 97(6), 2025.

[0224] Donegan et al., Effect of Ion-Pairing Reagent Hydrophobicity on Liquid Chromatographyand Mass Spectrometry Analysis of Oligonucleotides, Journal of Chromatography A, Vol.1666, 2022.

[0225] Roussis et al., Small Alkyl Amines as Ion-Pair Reagents for the Separation of PositionalIsomers of Impurities in Phosphate Diester oligonucleotides, Journal of Chromatography A, Vol. 1594, 2019.

[0226] Murugaiah et al., Reversed-Phase High-Performance Liquid Chromatography Method forSimultaneous Analysis of Two Liposome-Formulated Short Interfering RNA Duplexes, Analytical Biochemistry, Vol.401(1), 2010.

[0227] Li et al., Alkylamine Ion-Pairing Reagents and the Chromatographic Separation ofOligonucleotides, Journal of Chromatography A, Vol.1580, 2018.

[0228] Levin. et al., Combining Ion Pairing Agents for Enhanced Analysis of OligonucleotideTherapeutics by Reversed Phase-Ion Pairing Ultra Performance Liquid Chromatography (UPLC), Journal of Chromatography B, Vol.879(19), 2011.

[0229] Wochener et al., Method for Analyzing By-Products of RNA In Vitro Transcription,WO2017140345, 2017.

[0230] Niita et al., Method for Analyzing Nucleic Acid, Published US Patent Application No.20230116567, 2023.

[0231] Issa et al., Analytical HPLC Methods, US Patent No. 11,866,696, 2024.

[0232] Grobe et al., Method of Analyzing Contaminants in RNA products by Ion-PairChromatography, WO2024231285, 2024.

[0233] Duff et al., Methods for Separating Molecular Species of Guanine-Rich Oligonucleotides,Published Canadian Patent Application No.3232773, 2022.DOCKET NO.: 38120-6046 (11853WO01) EXAMPLES

[0234] Materials and Methods. The two siRNA molecules primarily described in thisdisclosure are referred to as siRNA-1 (or siRNA #1) and siRNA-2 (or siRNA #2). The siRNAs are composed of two complementary oligonucleotide strands (sense and antisense) that are hybridized, forming an alpha helical duplex conformation. siRNA-1 and siRNA-2 have the same length and a highly similar structure; however, siRNA-2 features a GNA nucleoside. The fixed dose combination product described in this disclosure contains siRNA-1 and siRNA-2 at a 1:1 mass ratio. The solutions tested in these examples contain 0.2 mg / mL siRNA-1 in water for injection (WFI), 0.2 mg / mL siRNA-2 in WFI, and 0.2 mg / mL of each siRNA-1 and siRNA-2 in a 1:1 siRNA-1:siRNA-2 mass ratio in WFI.

[0235] Subsequentially, another siRNA (siRNA-3), of the same size and similar structure, wasincorporated into two different fixed dose combinations containing siRNA-1 and siRNA-3 at a 1:1 mass ratio, and siRNA-2 and siRNA-3 at a 1:1 mass ratio. Both additional fixed dose combinations (siRNA-1: siRNA-3, siRNA-2: siRNA-3) tested were also formulated to contain 0.2 mg / mL of each siRNA in WFI.

[0236] An Acquity H-Class Ultra-Performance Liquid Chromatography system equipped with atunable ultraviolet (TUV) detector was used (Waters Corporation, Milford, MA). Acquity Premier Oligonucleotides BEH C18 (300 Å, 1.7 µm, 2.1 mm x 150 mm) and Acquity UPLC Peptide BEH C18 (300 Å, 1.7 µm, 2.1 mm x 100 mm) columns were used (Waters Corporation, Milford, MA). Reagents and solutions used include hexafluoro isopropanol (≥99% purity; HFIP), ethylenediaminetetraacetic acid (99.995% free acid in solid form; EDTA), triethylamine (≥99.5% purity; TEA), methanol, acetonitrile (LCMS grade), and Gibco Water for Injection (USP grade). Mobile Phase A consisted of HFIP (550 mM), TEA (13 mM), and EDTA (5 µM) in water / 10% methanol. Mobile Phase B1 consisted of EDTA (5 µM) in methanol, and Mobile Phase B2 consisted of EDTA (5 µM) and acetonitrile (4%) in methanol. Whenever just “mobile phase B” is mentioned herein, no acetonitrile was used. JMP®Version 17.1.1 software was used for data and statistical analysis. Example 1. Preliminary Method Assessment of Method Variables

[0237] siRNA-1 and siRNA-2 have highly similar structure and physicochemical properties.Separating these two similar siRNAs for routine testing is difficult using conventional methods. siRNA-1 and siRNA-2 were co-formulated at a 1:1 ratio. Given the similar chemical structure ofDOCKET NO.: 38120-6046 (11853WO01) siRNA-1 and siRNA-2 in the fixed dose combination, it was expected that separating the four mean peaks (antisense and sense strands of each siRNA) could be challenging.

[0238] An established method of denaturing IPRP-UPLC (d-IPRP-LC) was used for theseparation of the single strands of siRNA-1 and siRNA-2 in the fixed dose combination that had the following specification. An ACQUITY UPLC Peptide BEH C18 (300 Å, 1.7 μm, 2.1 mm x 100 mm) column was used at 80 °C (also referred to herein as medium column temperature). Mobile phase A was 550 mM HFIP, 13 mM TEA, 5 μM, EDTA, and water / 10% methanol. Mobile phase B was 5 μM EDTA in methanol. These conditions are also referred to herein as the “Base Method” (or also called the “Platform GalNAc Method”). This Base Method was characterized by the “Gradient A” as shown in FIG.1B.

[0239] This Base Method resulted in poor separation between the two AS (antisense strand)peaks, with a resolution of 0.6 (USP resolution), as shown in FIG.1A. For SS (sense strand) separation, a good resolution of 1.6 (USP resolution) was obtained. However, peak splitting was observed in the siRNA-1 SS.

[0240] A preliminary method assessment identified injection volume, mobile phase B gradient,column temperature, and mobile phase B composition as potential critical method variables. Preliminary studies were performed using the Base Method column (ACQUITY UPLC Peptide BEH C18 (300 Å, 1.7 μm, 2.1 mm x 100 mm) and Base Method mobile phases as the starting point, as shown in Table 2 below. The impact of each of the identified d-IPRP-LC parameters was assessed. The resulting peak shape showed major improvement based on the choice of gradient and lower injection volume yielded greater strand separation for both AS and SS strands, as shown in FIG.2A (gradient variation at 8 µL injection volume with peptide column), FIG.2B, FIG.2C, FIG.2D (gradient variation at 8 µL injection volume with peptide column), and FIG.2E (gradient variation at 5 µL injection volume with peptide column). Table 2: Method development using a peptide column, and varying gradient and injection volume Method B% B% Col. Inj. AS SS Peak Shape dDOCKET NO.: 38120-6046 (11853WO01) Method B% B% Col. Inj. AS SS Peak Shape T=0min T=40min Temp Vol. Resolution Resolution Annotation º * * d d d

[0241] Further d-IPRP-LC analyses were performed using an optimized column thatincorporates a high-performance surface technology for ultra-low non-specific adsorption (Acquity Premier Oligonucleotide BEH C18, 300 Å, 1.7 μm, 2.1 mm x 100 mm from Waters Corporation). The specific d-IPRP-LC parameters used are set forth in Table 3 below, and the resulting d-IPRP-LC chromatograms are shown in FIG.3A and FIG.3B. Overall, the oligonucleotide column used increased antisense strand separation and positively impacted peak shape. And lowering the injection volume and increasing the column temperature both positively impacted peak shape and AS peaks separation. Because the assessed parameters were found to be important, further statistical tools were employed to understand the impact of each parameter better.DOCKET NO.: 38120-6046 (11853WO01) Table 3: Method development using an oligonucleotides column (except for Base Method, where the peptide column was used), varying column temperature and injection volume Method B% B% Column Inj. AS SS Peak Shape T=0min T=40min Temp. Vol. Resolution Resolution Annotation d 1 f d f d s dExample 2. Design of Experiments study of single stranded oligonucleotide separation

[0242] Additional studies were conducted to systematically evaluate how different methodparameters, including injection volume, mobile phase B gradient, column temperature, and mobile phase B composition, affect the method performance, including peak resolution and peak shape, and further to identify optimal method conditions.

[0243] Design of Experiments (DoE) is a systematic approach to investigate a process ormethod, based on predefined goals targeting a limited number of experiments. Different factorsDOCKET NO.: 38120-6046 (11853WO01) are simultaneously evaluated, and the interaction between factors is also determined. The DoE study described in this disclosure included D-optimal design using 26 runs, as shown schematically in FIG.4. Responses included AS peak resolution, SS peak resolution, and overall peak shape.

[0244] All of the main factors previously identified, their interactions, and quadratics wereincluded in the model. In particular, the DoE study evaluated the following factors: Column temperature (75-85 °C), injection volume (2-8 µL), MP B% at T=0min (5-10%), MP B% at T=40min (22-27%), and mobile phase B type (with 4% acetonitrile or without acetonitrile).

[0245] The details and parameter specifics of the 26 D-optimal design runs are provided in Table4 below. Table 4: DoE D-Optimal Design for effect screening Column Injection DoE Temperature Volume Mobile Phase B Type Mobile Phase B Mobile Phase BDOCKET NO.: 38120-6046 (11853WO01) Note: Mobile Phase B base composition is 5µM EDTA in methanol.

[0246] Each DoE condition was collected thrice (3 injections) and the results obtained wereaveraged. The AS and SS strand peak resolution results were obtained using the Empower®3 Software that was part of the chromatography package used as described herein (Waters Corporation (Milford, MA)), but can also be calculated using the following equation:

[0247] An overall peak shape scoreon the scoring of the shape of eachindividual main peak. Peaks that did not present any splitting affecting its symmetry were not normalized and assigned a peak score of 2.5. Where peak splitting was observed, peaks were normalized before a peak score was calculated. This normalized peak score and the score of 2.5 for peaks without splitting was then used for the calculation of the final overall peak shape score.

[0248] A method for peak normalization is illustrated in FIG. 5A, FIG. 5B, FIG. 5C and FIG.5D. FIG.5A illustrates severe siRNA-1 SS peak splitting at 75 °C, and FIG.5B illustrates aspects of the process of peak normalization for the siRNA-1 SS strand peak. FIG.5C illustrates mild siRNA-1 AS peak splitting at 75 °C, and FIG.5D illustrates aspects of the process of peak normalization for the siRNA-1 AS strand peak.

[0249] Peak normalization values in case of peak splitting were calculated as follows: (Peak 1Height – Valley Height) / Peak 1 Height + (Split Peak Hight – Valley Height) / Split Peak Height.

[0250] Peak shape scores for normalized peaks were calculated as follows: Peak Score = ((PeakNormalization Maximum - Peak Normalization Result) / Peak Normalization Maximum) x Peak Maximum Score (2.5). The peak normalization maximum was the highest peak normalization value obtained from normalizing peaks from the same one of the four oligonucleotide stands across all experimental runs. Peak shape overall was expressed as sum peak score defined as the sum of each main peak score (AS siRNA-1, AS siRNA-2, SS siRNA-1, and SS siRNA-2) within one experimental run.

[0251] Peak normalization values for all 26 DoE runs (in triplicates) are shown in Table 5 below.AS siRNA-2 and SS siRNA-2 never displayed peak splitting and therefore always received a peak normalization value of “0”.DOCKET NO.: 38120-6046 (11853WO01) Table 5: Peak normalization values AS siRNA-1 AS siRNA-2 S SS siRNA-1 (max (max 0.13 S siRNA-2 DoE Result 5) 1.2)DOCKET NO.: 38120-6046 (11853WO01) 7439 0.12 0.00 0.00 0.13147442 0.12 0.00 0.00 0.13

[0252] Peak scores are shown in Table 6 below.DOCKET NO.: 38120-6046 (11853WO01) Table 6: Peak scores DoE ResultAS siRNA-1 AS siRNA-2 SS siRNA-2 SS siRNA-1 Peak ShapeID Peak Score OverallDOCKET NO.: 38120-6046 (11853WO01) 7443 0.02 2.50 2.50 2.20 7.207280 2.50 2.50 2.50 2.50 10.00

[0253] The measured responses for the 26 D-optimal design runs are provided in Table 7 below.DOCKET NO.: 38120-6046 (11853WO01) Table 7: Measured responses from DoE runs Responses DoSeparation Peak Scoree

[0254] The obtained data from the 26 runs were further analyzed statistically and used to buildthe DoE model, using the JMP®software package (Version 17.1.1). The results of the DoE study for AS strand peak resolution are shown in FIG.6A, FIG.6B and FIG.6C. The antisense strand resolution predicted by the DoE statistical model was well correlated with the antisense strand resolution results measured with an R²=0.98697 and a P value < 0.0001 (FIG.6A). Additionally, the residuals were evenly distributed, indicating a robust model fit (FIG.6B). The profiler of the effect of the terms in the DoE model on antisense strand resolution is shown in FIG.6C. The terms that show statistically significant and practically meaningful impact on antisense strandDOCKET NO.: 38120-6046 (11853WO01) resolution are highlighted in boxes in FIG.6D. The terms with a p-value ≤ 0.05 were considered having a statistically significant effect and the terms with a scaled estimate ≥ 5% of the total scaled estimates (or 0.0326) were considered having a practically meaningful effect.

[0255] The results of the DoE study for SS strand peak resolution are shown in FIG. 7A, FIG.7B and FIG.7C. The sense strand resolution predicted by the DoE statistical model was well correlated with the sense strand resolution results measured with an R²= 0.99671 and a P value < 0.0001 (FIG.7A). Additionally, the residuals were evenly distributed, indicating a robust model fit (FIG.7B). The profiler of the effect of the terms in the DoE model on sense strand resolution is shown in FIG.7C. The terms that show statistically significant and practically meaningful impact on sense strand resolution are highlighted in boxes in FIG.7D. The terms with a p-value ≤ 0.05 were considered having a statistically significant effect and the terms with a scaled estimate ≥ 5% of the total scaled estimates (or 0.0382) were considered having a practically meaningful effect.

[0256] The results of the DoE study for overall peak shape are shown in FIG. 8A, FIG. 8B andFIG.8C. The peak shape overall predicted by the DoE statistical model was well correlated with the peak shape results measured with an R²= 0.99108 and a P value < 0.0001 (FIG.8A). Additionally, the residuals were evenly distributed, indicating a robust model fit (FIG.8B). The profiler of the effect of the terms in the DoE model on overall peak shape is shown in FIG.8C. The terms that show statistically significant and practically meaningful impact on overall peak shape are highlighted in boxes in FIG.8D. The terms with a p-value ≤ 0.05 were considered having a statistically significant effect and the terms with a scaled estimate ≥ 5% of the total scaled estimates (or 0.1411) were considered having a practically meaningful effect. Example 3. Optimized d-IPRP-LC Conditions for Single-Stranded Oligonucleotide Separation

[0257] A graph profiler showing the trending of the impact of each factor on the response isshown in FIG.9. Using this profiler, optimal method conditions were identified. The optimal method condition was determined via maximizing the desirability function using the JMP®software (Version 17.1.1). The importance of the terms for maximizing the desirability function was set as antisense strand resolution = 0.5 and peak shape overall = 0.5. Sense strand resolutionDOCKET NO.: 38120-6046 (11853WO01) was excluded from the desirability analysis because good separation of the sense strands was achieved under most tested conditions. Table 8: Strategy for selecting an optimal method using a DoE study of d-IPRP-LC method parameters Output Response Goal ImportanceAS resolution Maximize 0.5Table10 below. Table 9: Optimal method conditions determined from DoE modeling Method Parameter Optimal dIPRP-LC method determined from DoE ModelingACQUITY Premier Oligonucleotide BEH C18 300 Å 1.7 μm 2.1Table 10: Optimal gradient determined from DoE modeling MP Gradient Ti i MP A MP B C

[0259] The performance of the DoE model was verified by actual experiments. The optimalconditions were validated using both the fixed dose combination and single siRNA solutions. The ability of the optimal assay to separate siRNA-1 and siRNA-2 AS and SS strands in the fixed dose combination was assessed (FIG.10B) and compared to the Base Method (FIG.10A).DOCKET NO.: 38120-6046 (11853WO01) Using the optimal method, the AS strands showed superior resolution and both AS and SS strand peaks showed improved peak shape compared to the Base Method (as shown and summarized in Table 11 below), demonstrating the usefulness of the optimal parameters. Table 11: Comparison of the Base Method and optimal method determined from DoE modeling dIPRP Method AS USP SS USP Resolution Resolution Peak Shape AnnotationndsiRNA-2 individually. Results are shown in FIG.11.

[0261] Assessment of the DoE study results yielded an optimal method employing the highestperforming attributes evaluated as having a statistically significant and practically meaningful impact to the antisense strand separation and peak shape attribute. The DoE model also highlights the parameters that have a positive impact on achieving high separation resolution and better peak shapes of single strands of the siRNA in the fixed dose combination, potentially speeding up future method development. Example 4. Analyses of Additional Fixed-Dose-Combinations (FDCs)

[0262] The optimal method was subsequently applied to two additional siRNA FDCs, includingeither siRNA-1 and siRNA-3 or siRNA-2 and siRNA-3. This experiment demonstrated superior method performance, as shown in FIG.12 and Table 12. Excellent peak shape and single strand separation were observed with the 3 siRNA-siRNA FDCs tested. Table 12: Resolution data from 3 different FDC products siRNA FDC AS USP Resolution SS USP Resolution Peak ShapeDOCKET NO.: 38120-6046 (11853WO01) siRNA FDC AS USP Resolution SS USP Resolution Peak Shape Annotation

Claims

DOCKET NO.: 38120-6046 (11853WO01) What is claimed is:

1. A method for separating at least two oligonucleotides in a sample, comprising: (a) obtaining a sample comprising at least two oligonucleotides; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotides, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

2. The method of claim 1, wherein said at least two oligonucleotides are sense strands of small interfering RNAs.

3. The method of claim 1, wherein said at least two oligonucleotides are antisense strands of small interfering RNAs.

4. The method of any one of claims 1 to 3, wherein said sample is a fixed dose combination of at least two small interfering RNAs.

5. The method of any one of claims 1 to 4, wherein said at least two oligonucleotides are detected using ultraviolet detection.

6. The method of any one of claims 1 to 5, wherein conditions for said separation further include a mobile phase A comprising hexafluoro isopropanol, triethylamine, and EDTA in water.

7. The method of any one of claims 1 to 6, wherein a total concentration of oligonucleotides in said sample is about 0.4 mg / mL.

8. The method of any one of claims 1 to 7, wherein a column for said denaturing ion pairing liquid chromatography separation is a C18 column.DOCKET NO.: 38120-6046 (11853WO01) 9. The method of any one of claims 1 to 8, wherein said column temperature is about 85 °C.

10. The method of any one of claims 1 to 9, wherein said injection volume is about 2 µL.

11. The method of any one of claims 1 to 10, wherein an initial concentration of said mobile phase B is about 5%.

12. The method of any one of claims 1 to 11, wherein a maximum concentration of said mobile phase B is about 22%.

13. The method of any one of claims 1 to 12, wherein a maximum concentration of said mobile phase B is at about 40 minutes.

14. A method for separating at least two oligonucleotide strands in a sample, comprising: (a) obtaining a sample comprising at least two oligonucleotide strands; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

15. The method of claim 14, wherein said at least two oligonucleotide strands are sense strands of small interfering RNAs.

16. The method of claim 14, wherein said at least two oligonucleotide strands are antisense strands of small interfering RNAs.

17. The method of any one of claims 14 to 16, wherein said sample is a fixed dose combination of at least two small interfering RNAs.DOCKET NO.: 38120-6046 (11853WO01) 18. The method of any one of claims 14 to 17, wherein said at least two oligonucleotide strands are detected using ultraviolet detection.

19. The method of any one of claims 14 to 18, wherein conditions for said separation further include a mobile phase A comprising hexafluoro isopropanol, triethylamine, and EDTA in water.

20. The method of any one of claims 14 to 19, wherein a total concentration of oligonucleotides in said sample is about 0.4 mg / mL.

21. The method of any one of claims 14 to 20, wherein a column for said denaturing ion pairing liquid chromatography separation is a C18 column.

22. The method of any one of claims 14 to 21, wherein said column temperature is about 85 °C.

23. The method of any one of claims 14 to 22, wherein said injection volume is about 2 µL.

24. The method of any one of claims 14 to 23, wherein an initial concentration of said mobile phase B is about 5%.

25. The method of any one of claims 14 to 24, wherein a maximum concentration of said mobile phase B is about 22%.

26. The method of any one of claims 14 to 25, wherein a maximum concentration of said mobile phase B is at about 40 minutes.

27. A method for separating a first single-stranded oligonucleotide from at least one additional single-stranded oligonucleotide, comprising: (a) obtaining a sample comprising a first single-stranded oligonucleotide and at least one additional single-stranded oligonucleotide; andDOCKET NO.: 38120-6046 (11853WO01) (b) subjecting said sample to denaturing ion pairing liquid chromatography separation, wherein conditions for said separation include a column temperature from about 80 °C to about 90 °C, an injection volume from about 1 µL to about 3 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 7% and a maximum concentration of said mobile phase B is from about 18% to about 26%.

28. The method of claim 27, wherein said first single-stranded oligonucleotide and said at least one additional single-stranded oligonucleotide are sense strands of small interfering RNAs.

29. The method of claim 27, wherein said first single-stranded oligonucleotide and said at least one additional single-stranded oligonucleotide are antisense strands of small interfering RNAs.

30. The method of any one of claims 27 to 29, wherein said sample is a fixed dose combination of at least two small interfering RNAs.

31. The method of any one of claims 27 to 30, wherein said single-stranded oligonucleotides are detected using ultraviolet detection.

32. The method of any one of claims 27 to 31, wherein conditions for said separation further include a mobile phase A comprising hexafluoro isopropanol, triethylamine, and EDTA in water.

33. The method of any one of claims 27 to 32, wherein a total concentration of oligonucleotides in said sample is about 0.4 mg / mL.

34. The method of any one of claims 27 to 33, wherein a column for said denaturing ion pairing liquid chromatography separation is a C18 column.

35. The method of any one of claims 27 to 34, wherein said column temperature is about 85 °C.DOCKET NO.: 38120-6046 (11853WO01) 36. The method of any one of claims 27 to 35, wherein said injection volume is about 2 µL.

37. The method of any one of claims 27 to 36, wherein an initial concentration of said mobile phase B is about 5%.

38. The method of any one of claims 27 to 37, wherein a maximum concentration of said mobile phase B is about 22%.

39. The method of any one of claims 27 to 38, wherein a maximum concentration of said mobile phase B is at about 40 minutes.

40. A method for separating at least two oligonucleotide strands in a sample, comprising: (a) obtaining a sample comprising at least two oligonucleotide strands; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%.

41. The method of claim 40, wherein said at least two oligonucleotide strands are antisense strands of small interfering RNAs.

42. The method of claim 41, wherein said sample is a fixed dose combination of at least two small interfering RNAs.

43. A method for separating four oligonucleotide strands in a sample, comprising: (a) obtaining a sample comprising four oligonucleotide strands; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include aDOCKET NO.: 38120-6046 (11853WO01) column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%; wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in the sample not included in said other small interfering RNA.

44. The method of claim 43, wherein each of said two small interfering RNAs comprises an oligonucleotide strand that is connected to a non-oligonucleotide moiety.

45. The method of claim 44, wherein said non-oligonucleotide moiety comprises a GalNAc moiety.

46. The method of claim 45, wherein each of said four oligonucleotide strands comprises a phosphorothioate (PS) backbone modification.

47. A method for separating four oligonucleotide strands in a sample, comprising: (a) obtaining a sample comprising four oligonucleotide strands; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of about 85 °C, an injection volume of about 2 µL, a mobile phase B comprising about 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at about T=0min an initial concentration of said mobile phase B is about 5% and at about T=40min a concentration of said mobile phase B is about 22%; wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA; wherein each of said two small interfering RNAs comprises an oligonucleotide strand that is connected to a non-oligonucleotide moiety that comprises a GalNAc moiety; andDOCKET NO.: 38120-6046 (11853WO01) wherein each of said four oligonucleotide strands comprises a phosphorothioate (PS) backbone modification.

48. A method for separating four oligonucleotide strands in a sample, comprising: (a) obtaining a sample comprising four oligonucleotide strands; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said four oligonucleotide strands, wherein conditions for said separation include a column temperature of 85 °C, an injection volume of 2 µL, a mobile phase B comprising 5 µM ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein at T=0min an initial concentration of said mobile phase B is 5% and at T=40min a concentration of said mobile phase B is 22%; wherein said sample is a fixed dose combination of two small interfering RNAs each consisting of two of said four oligonucleotide strands in said sample not included in said other small interfering RNA; wherein each of said two small interfering RNAs comprises an oligonucleotide strand that is connected to a non-oligonucleotide moiety that comprises a GalNAc moiety; and wherein each of said four oligonucleotide strands comprises a phosphorothioate (PS) backbone modification.

49. A method for separating at least two oligonucleotides in a sample, comprising: (a) obtaining a sample comprising at least two oligonucleotides; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotides, wherein conditions for said separation include a column temperature from about 75 °C to about 90 °C, an injection volume from about 1 µL to about 8 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 10% and a maximum concentration of said mobile phase B is from about 18% to about 27%.

50. The method of claim 49, wherein said at least two oligonucleotides are sense strands of small interfering RNAs.DOCKET NO.: 38120-6046 (11853WO01) 51. The method of claim 49, wherein said at least two oligonucleotides are antisense strands of small interfering RNAs.

52. The method of any one of claims 49 to 51, wherein said sample is a fixed dose combination of at least two small interfering RNAs.

53. The method of any one of claims 49 to 52, wherein said at least two oligonucleotides are detected using ultraviolet detection.

54. The method of any one of claims 49 to 53, wherein conditions for said separation further include a mobile phase A comprising hexafluoro isopropanol, triethylamine, and EDTA in water.

55. The method of any one of claims 49 to 54, wherein a total concentration of oligonucleotides in said sample is about 0.4 mg / mL.

56. The method of any one of claims 49 to 55, wherein a column for said denaturing ion pairing liquid chromatography separation is a C18 column.

57. The method of any one of claims 49 to 56, wherein said column temperature is about 85 °C.

58. The method of any one of claims 49 to 57, wherein said injection volume is about 2 µL.

59. The method of any one of claims 49 to 58, wherein an initial concentration of said mobile phase B is about 5%.

60. The method of any one of claims 49 to 59, wherein a maximum concentration of said mobile phase B is about 22%.

61. The method of any one of claims 49 to 60, wherein a maximum concentration of said mobile phase B is at about 40 minutes.DOCKET NO.: 38120-6046 (11853WO01) 62. A method for separating at least two oligonucleotide strands in a sample, comprising: (a) obtaining a sample comprising at least two oligonucleotide strands; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation to separate said at least two oligonucleotide strands, wherein conditions for said separation include a column temperature from about 75 °C to about 90 °C, an injection volume from about 1 µL to about 8 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 10% and a maximum concentration of said mobile phase B is from about 18% to about 27%.

63. The method of claim 62, wherein said at least two oligonucleotide strands are sense strands of small interfering RNAs.

64. The method of claim 62, wherein said at least two oligonucleotide strands are antisense strands of small interfering RNAs.

65. The method of any one of claims 62 to 64, wherein said sample is a fixed dose combination of at least two small interfering RNAs.

66. The method of any one of claims 62 to 65, wherein said at least two oligonucleotide strands are detected using ultraviolet detection.

67. The method of any one of claims 62 to 66, wherein conditions for said separation further include a mobile phase A comprising hexafluoro isopropanol, triethylamine, and EDTA in water.

68. The method of any one of claims 62 to 67, wherein a total concentration of oligonucleotides in said sample is about 0.4 mg / mL.

69. The method of any one of claims 62 to 68, wherein a column for said denaturing ion pairing liquid chromatography separation is a C18 column.DOCKET NO.: 38120-6046 (11853WO01) 70. The method of any one of claims 62 to 69, wherein said column temperature is about 85 °C.

71. The method of any one of claims 62 to 70, wherein said injection volume is about 2 µL.

72. The method of any one of claims 62 to 71, wherein an initial concentration of said mobile phase B is about 5%.

73. The method of any one of claims 62 to 72, wherein a maximum concentration of said mobile phase B is about 22%.

74. The method of any one of claims 62 to 73, wherein a maximum concentration of said mobile phase B is at about 40 minutes.

75. A method for separating a first single-stranded oligonucleotide from at least one additional single-stranded oligonucleotide, comprising: (a) obtaining a sample comprising a first single-stranded oligonucleotide and at least one additional single-stranded oligonucleotide; and (b) subjecting said sample to denaturing ion pairing liquid chromatography separation, wherein conditions for said separation include a column temperature from about 75 °C to about 90 °C, an injection volume from about 1 µL to about 8 µL, a mobile phase B comprising ethylenediaminetetraacetic acid (EDTA) in methanol, and a gradient wherein an initial concentration of said mobile phase B is from about 3% to about 10% and a maximum concentration of said mobile phase B is from about 18% to about 27%.

76. The method of claim 75, wherein said first single-stranded oligonucleotide and said at least one additional single-stranded oligonucleotide are sense strands of small interfering RNAs.

77. The method of claim 75, wherein said first single-stranded oligonucleotide and said at least one additional single-stranded oligonucleotide are antisense strands of small interfering RNAs.DOCKET NO.: 38120-6046 (11853WO01) 78. The method of any one of claims 75 to 77, wherein said sample is a fixed dose combination of at least two small interfering RNAs.

79. The method of any one of claims 75 to 78, wherein said single-stranded oligonucleotides are detected using ultraviolet detection.

80. The method of any one of claims 75 to 79, wherein conditions for said separation further include a mobile phase A comprising hexafluoro isopropanol, triethylamine, and EDTA in water.

81. The method of any one of claims 2775 to 80, wherein a total concentration of oligonucleotides in said sample is about 0.4 mg / mL.

82. The method of any one of claims 75 to 81, wherein a column for said denaturing ion pairing liquid chromatography separation is a C18 column.

83. The method of any one of claims 75 to 82, wherein said column temperature is about 85 °C.

84. The method of any one of claims 75 to 83, wherein said injection volume is about 2 µL.

85. The method of any one of claims 75 to 84, wherein an initial concentration of said mobile phase B is about 5%.

86. The method of any one of claims 75 to 85, wherein a maximum concentration of said mobile phase B is about 22%.

87. The method of any one of claims 75 to 86, wherein a maximum concentration of said mobile phase B is at about 40 minutes.

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