Purification methods for carbohydrate-linked oligonucleotides

Abstract

To provide a novel preparative purification method for nucleic acid therapeutics such as carbohydrate-conjugated oligonucleotides.SOLUTION: The present invention relates to methods for purifying carbohydrate-conjugated oligonucleotides using a mixed-mode stationary phase and a mobile phase comprising a dual salt / organic solvent gradient. Methods for purifying carbohydrate-conjugated oligonucleotides using an anion exchange stationary phase and a mobile phase comprising a dual pH / salt gradient are also described.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 866,515, filed June 25, 2019, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files

[0001] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on June 24, 2020, is titled A-2362-WO-PCT_SeqList_ST25, and is 13 kilobytes in size.

[0003] The present invention relates to the technical field of nucleic acid purification. In particular, the present invention relates to a method for purifying carbohydrate-oligonucleotide conjugate compounds using mixed-mode chromatography. The method allows for the purification of intact carbohydrate-oligonucleotide conjugate compounds from unconjugated oligonucleotides and other impurities. The method also allows for the separation of phosphorothioate diastereomers of oligonucleotides containing one or more phosphorothioate internucleotide linkages. The present invention also relates to a method for purifying carbohydrate-oligonucleotide conjugate compounds using an anion exchange stationary phase and elution with a dual pH / salt gradient. Such a method can be used in combination with the mixed-mode chromatography method described herein to purify carbohydrate-oligonucleotide conjugate compounds. [Background technology]

[0004] Progress continues to be made in the development of nucleic acid-based therapeutics with gene silencing mechanisms of action.However, one important challenge to the development of this class of therapeutic molecules is the difficulty of targeting therapeutic nucleic acids to the appropriate tissue or cells.One approach to deliver nucleic acid molecules to liver cells is to conjugate therapeutic nucleic acids to carbohydrate molecules that bind to receptors such as asialoglycoprotein receptors on the surface of liver cells.These and other chemical modifications to nucleic acid structure, which facilitate delivery and enhance in vivo efficacy and stability, require the development of new purification methods for these increasingly complex molecules.

[0005] Both ion exchange and reverse-phase liquid chromatography have been used previously to purify natural and synthetic oligonucleotides. Reverse-phase chromatography methods generally require the use of a 5'-protecting group, such as 5'-O-trityl, to protect the 5' hydroxyl group during oligonucleotide synthesis and then purify the full-length oligonucleotide sequence ("trityl-on" sequence) from shortened failure sequences lacking the protecting group ("trityl-off" sequence). These reverse-phase methods often require an additional step after purification to remove the protecting group, thereby increasing costs and overall processing time. Chromatographic purification methods based on ion exchange are generally less costly than reverse-phase methods because they use aqueous-based mobile phases. However, structural modifications made to current nucleic acid therapeutics to enable in vivo use increase the complexity of these molecules and the types of impurities generated during their synthesis, so traditional ion exchange and reverse-phase chromatography methods are not always suitable for achieving the required purity and yield. Therefore, there is a need in the art for novel preparative purification methods for nucleic acid therapeutics, such as carbohydrate-conjugated oligonucleotides. Summary of the Invention [Means for solving the problem]

[0006] The present invention is based, in part, on the development of an orthogonal separation method to conventional anion-exchange chromatography methods for oligonucleotides, particularly carbohydrate-conjugated oligonucleotides. In certain embodiments, the methods of the present invention utilize a mixed-mode stationary phase containing strong anion-exchange ligands, strong cation-exchange ligands, and hydrophobic ligands (e.g., alkyl chains) combined with a tailored mobile phase that takes full advantage of both ion-exchange and hydrophobic interactions to control the retention and separation of carbohydrate-conjugated oligonucleotides from impurities, including unconjugated oligonucleotides and failure sequences.

[0007] Thus, the present invention provides a method for purifying a carbohydrate-oligonucleotide conjugate compound from one or more impurities. In one embodiment, the method comprises contacting a solution containing the carbohydrate-oligonucleotide conjugate compound and one or more impurities with a mixed-mode matrix; passing a mobile phase described herein through the mixed-mode matrix; collecting elution fractions from the mixed-mode matrix, wherein the one or more impurities are eluted in a first set of elution fractions and the carbohydrate-oligonucleotide conjugate compound is eluted in a second set of elution fractions, thereby separating the carbohydrate-oligonucleotide conjugate compound from the impurities.

[0008] The mixed-mode matrix used in the methods of the present invention is generally composed of ligands having positively charged functional groups, negatively charged functional groups, and hydrophobic functional groups. In certain embodiments, the mixed-mode matrix includes strong anion-exchange ligands, strong cation-exchange ligands, and hydrophobic ligands. The strong anion-exchange ligands and strong cation-exchange ligands remain fully charged over a wide pH range and exhibit little or no change in ion-exchange capacity with changes in pH. In some embodiments, the strong anion-exchange ligands include quaternary amines. In these and other embodiments, the strong cation-exchange ligands include sulfonyl functional groups. The hydrophobic ligands in the mixed-mode matrix can include alkyl groups (e.g., isopropyl, propyl, t-butyl, butyl, and C8-C18 alkyl chains) or aryl groups (e.g., phenyl groups). In certain embodiments, the hydrophobic ligands include alkyl groups. In one embodiment, the hydrophobic ligands include octadecyl carbon chains (e.g., C18 alkyl chains). In another embodiment, the hydrophobic ligands include octyl carbon chains (e.g., C8 alkyl chains). The mixed mode matrix used in the methods of the present invention may have a pore size of less than about 20 nm, for example, from about 8 nm to about 15 nm or from about 11 nm to about 14 nm.

[0009] The mobile phase used in the mixed-mode chromatography method of the present invention has a pH of about 7.0 to about 8.5 and comprises a buffer and an organic solvent. The buffer can be any buffer capable of maintaining a pH in the target range, such as sodium phosphate, Tris hydrochloride, HEPES, or MOPS, and can be present at a concentration of about 20 mM to about 200 mM. In one embodiment, the mobile phase comprises about 80 mM to about 110 mM of buffer. Suitable organic solvents that can be used in the mobile phase include acetonitrile, methanol, propanol, isopropanol, ethanol, butanol, tetrahydrofuran, or acetone. The concentration of the organic solvent in the mobile phase can be increased over the course of the separation. For example, in some embodiments, the increasing concentration of the organic solvent in the mobile phase is a concentration gradient, e.g., from about 8% (v / v) to about 20% (v / v), from about 10% (v / v) to about 18% (v / v), from about 9% (v / v) to about 16% (v / v), or from about 11% (v / v) to about 17% (v / v). The concentration gradient can be a linear gradient or a step gradient. In certain embodiments, the concentration of the organic solvent in the mobile phase at the start of the separation is at least 8% (v / v) and increases over the course of the separation. In other embodiments, the concentration of the organic solvent in the mobile phase at the start of the separation is at least 10% (v / v) and increases over the course of the separation.

[0010] The mobile phase used in the mixed-mode chromatography of the present invention also contains an eluting salt, the concentration of which increases over the course of the separation. The eluting salt can be, for example, sodium, potassium, ammonium, trimethylammonium, triethylammonium, chloride, bromide, nitrate, nitrite, iodide, perchlorate, acetate, or formate. In certain embodiments, the eluting salt is sodium bromide, potassium bromide, ammonium bromide, sodium chloride, potassium chloride, or ammonium chloride. The increasing concentration of the eluting salt in the mobile phase during the separation can be, for example, a concentration gradient of eluting salt from 0 M to 1 M or from about 0.5 M to 1 M. The concentration gradient can be a linear gradient or a step gradient. In some embodiments, the mobile phase has a pH of about 7.0 to about 8.0 and comprises Tris-hydrochloride buffer, acetonitrile, and sodium bromide, and the concentration of sodium bromide increases in a gradient from about 0.5 M to about 1 M over the course of the separation. In such embodiments, the concentration of acetonitrile in the mobile phase may be increased in a gradient from about 8% (v / v) to about 20% (v / v) over the course of the separation.

[0011] Another aspect of the present invention relates to the development of an improved anion exchange chromatography-based method for purifying carbohydrate-oligonucleotide conjugate compounds. This improved method uses an anion exchange stationary phase containing a strong anion exchange ligand and a mobile phase containing both an increasing pH and an increasing salt gradient. Thus, in certain embodiments, the present invention provides a method for purifying carbohydrate-oligonucleotide conjugate compounds from one or more impurities, comprising contacting an anion exchange matrix with a solution containing the carbohydrate-oligonucleotide conjugate compound and one or more impurities; passing a mobile phase described herein through the anion exchange matrix; and collecting elution fractions from the anion exchange matrix, wherein the carbohydrate-oligonucleotide conjugate compound is eluted in a first set of elution fractions and one or more impurities are eluted in a second set of elution fractions, thereby separating the carbohydrate-oligonucleotide conjugate compound from the impurities.

[0012] The anion exchange matrix used in the methods of the present invention comprises a ligand having a positively charged functional group. Preferably, the anion exchange matrix comprises a strong anion exchange ligand that exhibits little or no change in ion exchange capacity with pH and remains positively charged over a wide pH range. In some embodiments, the strong anion exchange ligand comprises a quaternary amine, such as quaternary aminoethyl, quaternary ammonium, and quaternary aminomethyl.

[0013] The mobile phase used in the anion exchange chromatography method of the present invention comprises a buffer and an organic solvent. The organic solvent can be any of those described herein as suitable for use with the mobile phase for the mixed-mode chromatography method of the present invention, such as acetonitrile, methanol, propanol, isopropanol, ethanol, butanol, tetrahydrofuran, or acetone. In some embodiments, the organic solvent can be present in the mobile phase for the anion exchange chromatography method of the present invention at a concentration of about 1% (v / v) to about 50% (v / v), or about 1% (v / v) to about 20% (v / v). The pH of the mobile phase for the anion exchange chromatography method is generally at least about 8.5 and increases over the course of the separation. For example, in some embodiments, the pH of the mobile phase increases from about 8.5 to about 11 over the course of the separation. In other embodiments, the pH of the mobile phase increases from about 9.0 to about 10.5 over the course of the separation. The buffer can be any buffer capable of maintaining the pH of the mobile phase over the range of the pH gradient. One particularly suitable buffer across the range of the pH gradient is sodium phosphate.

[0014] The mobile phase for the anion exchange chromatography method of the present invention also includes an eluting salt, the concentration of which increases over the course of the separation. The eluting salt included in the mobile phase can be any of the eluting salts described herein for use with mobile phases for the mixed-mode chromatography method of the present invention. For example, in certain embodiments, the eluting salt is sodium bromide, potassium bromide, ammonium bromide, sodium chloride, potassium chloride, or ammonium chloride. In certain embodiments, the eluting salt is sodium chloride. The increasing concentration of the eluting salt in the mobile phase can be a concentration gradient (e.g., a linear gradient or a step gradient) of the eluting salt, for example, from 0 M to about 2 M or from 0 M to about 1 M.

[0015] The mobile phase for the anion exchange chromatography method of the present invention preferably comprises a dual pH / salt gradient—i.e., both the pH of the mobile phase and the elution salt concentration in the mobile phase are increased over the course of the separation. In some embodiments, the mobile phase comprises a pH gradient of about 8.5 to about 11 and an elution salt gradient of about 0 M to about 1 M. In other embodiments, the mobile phase comprises a pH gradient of about 9.0 to about 10.5 and an elution salt gradient of about 0.3 M to about 0.7 M. In yet other embodiments, the mobile phase comprises a pH gradient of about 8.5 to about 10.5 and an elution salt gradient of about 0 M to about 0.8 M. In certain embodiments, the mobile phase for the anion exchange chromatography method of the present invention comprises sodium phosphate buffer, acetonitrile, and sodium chloride, and the concentration of sodium chloride is increased at a gradient of about 0 M to about 1 M over the course of the separation, and the pH of the mobile phase is increased from about pH 8.5 to about 11.

[0016] In certain embodiments of the mixed-mode and anion-exchange chromatography methods of the present invention, the method further comprises isolating an elution fraction or set of elution fractions containing the carbohydrate-oligonucleotide conjugate compound. The isolated elution fractions may be subjected to one or more further processing steps, such as one or more further purification steps (e.g., desalting), an annealing reaction to hybridize the carbohydrate-oligonucleotide conjugate compound with a complementary strand to form a double-stranded RNA interference agent, and a formulation step to prepare a pharmaceutical composition of the carbohydrate-oligonucleotide conjugate compound for administration to a patient for therapeutic purposes. The mixed-mode chromatography methods of the present invention may be used in combination with the anion-exchange chromatography methods of the present invention to purify the carbohydrate-oligonucleotide conjugate compound. In some embodiments, the anion-exchange chromatography method of the present invention is performed first, followed by the mixed-mode chromatography method. In other embodiments, the mixed-mode chromatography method of the present invention is performed first, followed by the anion-exchange chromatography method.

[0017] The oligonucleotide component of a carbohydrate-oligonucleotide conjugate compound that can be purified by the methods of the present invention can be a natural or synthetic oligonucleotide. In some embodiments, the oligonucleotide component of a carbohydrate-oligonucleotide conjugate compound is a therapeutic oligonucleotide designed to target a gene or RNA molecule associated with a disease or disorder. Such therapeutic oligonucleotides include small hairpin RNAs (shRNAs), precursor miRNAs (pre-miRNAs), anti-miRNA oligonucleotides (e.g., antagomirs and antimiRs), antisense oligonucleotides, small interfering RNAs (siRNAs), microRNAs (miRNAs), or miRNA mimics.

[0018] The carbohydrate component of a carbohydrate-oligonucleotide conjugate compound that can be purified by the methods of the present invention can contain one or more hexose or hexosamine units, such as galactose, galactosamine, or N-acetyl-galactosamine. In certain embodiments, the carbohydrate component of the carbohydrate-oligonucleotide conjugate compound contains a multivalent galactose or N-acetyl-galactosamine moiety. Such multivalent sugar moieties can be trivalent or tetravalent. The carbohydrate-oligonucleotide conjugate compound that can be purified according to the methods of the present invention can contain one or more modified nucleotides, such as 2'-modified nucleotides. In some embodiments, the carbohydrate-oligonucleotide conjugate compound contains at least one phosphorothioate internucleotide linkage. The incorporation of a phosphorothioate internucleotide linkage generates diastereomers of the carbohydrate-oligonucleotide conjugate compound. In some embodiments, the mixed-mode chromatography method of the present invention provides for the separation of different sets of such phosphorothioate diastereomers. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows a preparative chromatogram of the purification of four GalNAc-conjugated oligonucleotides (compound numbers 47-04, 40-07, 40-04, and 40-01) using a polymeric bead-based anion-exchange resin (TSK-gel SuperQ-5PW column). Solutions containing each GalNAc-conjugated oligonucleotide were separated on a TSK-gel SuperQ-5PW column (21.5 × 150 mm, 13 μm) using a 20 mM NaHPO, 10% acetonitrile (v / v) mobile phase, pH 8.5, at a flow rate of 8 mL / min, eluting with an increasing gradient of sodium bromide. Detection was by UV absorption at 260 nm. Box 1 shows the peak corresponding to the intact GalNAc-conjugated oligonucleotide, while Box 2 highlights the peak corresponding to the unconjugated oligonucleotide. Box 3 encompasses peaks corresponding to higher-order structures of the oligonucleotide resulting from secondary interactions. [Figure 2]Figure 2 shows the separation of two GalNAc-conjugated oligonucleotides (compound nos. 40-01 (trace A) and 09-01 (trace B)) using a mixed-mode stationary phase (Scherzo SS-C18 column). Solutions containing each GalNAc-conjugated oligonucleotide were separated on a Scherzo SS-C18 column (4.6 x 50 mm, 3 μm) at a flow rate of 1.5 mL / min. The mobile phase consisted of 100 mM Tris, pH 7.5 (mobile phase A) and 1 M NaBr in 100 mM Tris, 20% (v / v) acetonitrile, pH 7.5 (mobile phase B). The gradient conditions were 40–70% mobile phase B from 0–20 min, followed by 40% mobile phase B at 20.1 min (5 min hold). Detection was by UV absorbance at 260 nm. [Figure 3] Figure 3 shows the purification of GalNAc-conjugated oligonucleotides (compound no. 34-01) using either a mixed-mode stationary phase (Scherzo SS-C18 column; trace I) or an anion-exchange stationary phase (TSK-gel SuperQ-5PW column; trace II). For the separation shown in trace I, a solution containing GalNAc-conjugated oligonucleotides was separated on a Scherzo SS-C18 column (10 × 250 mm, 3 μm) at a flow rate of 5 mL / min. The mobile phase consisted of 100 mM Tris, pH 7.5 (mobile phase A) and 1 M NaBr in 100 mM Tris, 20% (v / v) acetonitrile, pH 7.5 (mobile phase B). The gradient conditions were 55–80% mobile phase B from 0–40 min, 80% mobile phase B from 40–50 min, and 55% mobile phase B from 50.1–70 min. For the separation shown in Trace II, a solution containing GalNAc-conjugated oligonucleotides was separated on a TSK-gel SuperQ-5PW column (21.5 × 300 mm, 13 μm) using a 20 mM NaHPO, 15% acetonitrile (v / v) mobile phase, pH 8.5, at a flow rate of 8.5 mL / min and eluted with a pH / salt gradient. Detection was by UV absorbance at 260 nm. The dotted box indicates the fractions collected for further analysis. [Figure 4A]Figure 4A shows a preparative chromatogram of the separation of various GalNAc-conjugated oligonucleotides using a mixed-mode stationary phase (Scherzo SS-C18 column). Solutions containing each GalNAc-conjugated oligonucleotide were separated on a Scherzo SS-C18 column (10 × 250 mm, 3 μm) at a flow rate of 5 mL / min. The mobile phase consisted of 100 mM Tris, pH 7.5 (mobile phase A) and 1 M NaBr in 100 mM Tris, 20% (v / v) acetonitrile, pH 7.5 (mobile phase B). The gradient conditions were: 55–80% mobile phase B from 0–40 min, 80% mobile phase B from 40–50 min, and 55% mobile phase B from 50.1–70 min. Traces A to G correspond to the separations for compound numbers 13-10, 13-13, 13-07, 32-10, 32-07, 32-04, and 32-01, respectively. Detection was by UV absorbance at 260 nm. Dotted boxes represent fractions collected for further analysis. [Figure 4B] Figure 4B shows ion-pairing reversed-phase liquid chromatograms of the collected fractions from the preparative mixed-mode chromatographic separation shown in Figure 4A. The collected fractions containing each of the GalNAc-conjugated oligonucleotides (indicated by the dotted box in Figure 4A) were combined, desalted, and analyzed by ion-pairing reversed-phase liquid chromatography using a Waters Xbridge BEH OST C18 column (2.1 x 50 mm, 1.7 μm) and a 15.7 mM N,N-diisopropylethylamine (DIEA), 50 mM hexafluoro-2-propanol mobile phase with acetonitrile gradient elution. Traces A–G correspond to the separations for compounds 13-10, 13-13, 13-07, 32-10, 32-07, 32-04, and 32-01, respectively. Detection at 260 nm absorbance. For visualization, each of traces B to G was shifted 0.3 min (x-axis) and 1e+6 (y-axis) from the previous trace. The major peak in each trace had approximately the same retention time. [Figure 5]Figure 5 shows preparative chromatograms for two GalNAc-conjugated oligonucleotides (compound numbers 19-04 (trace A) and 19-07 (trace B)) using a mixed-mode stationary phase (Scherzo SS-C18 column). Solutions containing each GalNAc-conjugated oligonucleotide were separated on a Scherzo SS-C18 column (10 × 250 mm, 3 μm) at a flow rate of 5 mL / min. The mobile phase consisted of 100 mM Tris, pH 7.5 (mobile phase A) and 1 M NaBr in 100 mM Tris, pH 7.5, 20% (v / v) acetonitrile (mobile phase B). The gradient conditions were: 45–70% mobile phase B from 0–40 min, 70–80% mobile phase B from 40–45 min, 80% mobile phase B from 45–50 min, and 45% mobile phase B at 51 min (15 min hold). Detection was by UV absorbance at 260 nm. The dotted boxes correspond to the fractions collected for further analysis. [Figure 6] Figure 6 shows analytical chromatograms for the separation of a solution containing an oligonucleotide (compound no. 08-17) containing four phosphorothioate internucleotide linkages using either an anion-exchange stationary phase (TSK-gel SuperQ-5PW column; trace A) or a mixed-mode stationary phase (Scherzo SS-C18 column; trace B). For the separation shown in trace A, the solution containing the oligonucleotide was separated on a TSK-gel SuperQ-5PW column (7.5 × 75 mm, 10 μm) using a 20 mM NaHPO, 15% acetonitrile (v / v) mobile phase, pH 8.5, at a flow rate of 2 mL / min, eluting with a pH / salt gradient. For the separation shown in trace B, the solution containing the oligonucleotide was separated on a Scherzo SS-C18 column (4.6 × 50 mm, 3 μm) at a flow rate of 1 mL / min. The mobile phase consisted of 100 mM Tris, pH 7.5 (mobile phase A) and 1 M NaBr in 100 mM Tris, 20% (v / v) acetonitrile, pH 7.5 (mobile phase B). The gradient conditions were 55–80% mobile phase B from 0–8 min, 80% mobile phase B from 8–10 min, and 55% mobile phase B from 10.1–12 min. Detection was by UV absorbance at 260 nm. [Figure 7A]Figure 7A shows a preparative chromatogram of the separation of a solution containing an oligonucleotide (compound no. 08-17) containing four phosphorothioate internucleotide linkages using a mixed-mode stationary phase (Scherzo SS-C18 column). The solution containing the oligonucleotide was separated on a Scherzo SS-C18 column (10 x 250 mm, 3 µm) at a flow rate of 5 mL / min using a mixed-mode stationary phase (Scherzo SS-C18 column). The mobile phase consisted of 100 mM Tris, pH 7.5 (mobile phase A) and 1 M NaBr in 100 mM Tris, 20% (v / v) acetonitrile, pH 7.5 (mobile phase B). The gradient conditions were: 45–70% mobile phase B from 0–40 min, 70–80% mobile phase B from 40–45 min, 80% mobile phase B from 45–50 min, and 45% mobile phase B at 51 min (15 min hold). The peaks labeled 1, 2, and 3 were collected as individual fractions for further analysis. Detection was by UV absorbance at 260 nm. [Figure 7B] Figure 7B shows an ion-pairing reversed-phase liquid chromatogram of collected fractions from the preparative mixed-mode chromatographic separation shown in Figure 7A. The peaks labeled 1, 2, and 3 in Figure 7A were collected as individual fractions, desalted, and analyzed by ion-pairing reversed-phase liquid chromatography using a Waters Xbridge BEH OST C18 column (2.1 x 50 mm, 1.7 μm) and a 15.7 mM DIEA, 50 mM hexafluoro-2-propanol mobile phase with acetonitrile gradient elution. Purity values ​​are provided above for each trace. Detection was at 260 nm absorbance. For visualization purposes, traces 2 and 3 have each been shifted from the previous trace on both the x- and y-axes. [Figure 8A]Figure 8A shows a preparative chromatogram of the separation of various GalNAc-conjugated oligonucleotides using a mixed-mode stationary phase (Scherzo SS-C18 column). Solutions containing each GalNAc-conjugated oligonucleotide were separated on a Scherzo SS-C18 column (10 × 250 mm, 3 μm) at a flow rate of 5 mL / min. The mobile phase consisted of 100 mM Tris, pH 7.5 (mobile phase A) and 1 M NaBr in 100 mM Tris, 20% (v / v) acetonitrile, pH 7.5 (mobile phase B). The gradient conditions were 55–80% mobile phase B from 0–40 min, 80% mobile phase B from 40–50 min, and 55% mobile phase B from 50.1–70 min. Traces A–F correspond to the separations for compounds 24-10, 24-13, 24-16, 24-19, 24-22, and 24-25, respectively. Detection was by UV absorbance at 260 nm. The dotted boxes correspond to the fractions collected for further analysis. [Figure 8B] Figure 8B shows an ion-pairing reversed-phase liquid chromatogram of the collected fractions from the preparative mixed-mode chromatographic separation shown in Figure 8A. The collected fractions containing each of the GalNAc-conjugated oligonucleotides (indicated by the dotted box in Figure 8A) were combined, desalted, and analyzed by ion-pairing reversed-phase liquid chromatography using a Waters Xbridge BEH OST C18 column (2.1 x 50 mm, 1.7 μm) and a 15.7 mM DIEA, 50 mM hexafluoro-2-propanol mobile phase, eluted with an acetonitrile gradient. Traces A–F correspond to the separations for compounds 24-10, 24-13, 24-16, 24-19, 24-22, and 24-25, respectively. Detection was at 260 nm absorbance. For visualization, each of traces B–F was offset 0.5 min (x-axis) and 2e+5 (y-axis) from the previous trace. [Figure 9A]Figure 9A shows chromatograms of the separation of GalNAc-conjugated oligonucleotides (compound No. 34-01) using an anion-exchange stationary phase (TSK-gel SuperQ-5PW column; 7.5 × 75 mm, 10 μm) and eluting with either a dual pH / salt gradient (trace A) or a salt gradient (trace B). For the separation shown in trace A, a solution containing GalNAc-conjugated oligonucleotides was separated on the column using a 20 mM NaHPO, 10% acetonitrile (v / v) mobile phase at a flow rate of 2 mL / min, eluting with sodium bromide and an increasing gradient from pH 8.5 to 11. For the separation shown in trace B, a solution containing GalNAc-conjugated oligonucleotides was separated on the column using a 20 mM NaHPO, 10% acetonitrile (v / v) mobile phase at pH 8.5, eluting with an increasing gradient of sodium bromide and pH 8.5 to 11, eluting with a flow rate of 2 mL / min. Separations were performed at 40°C. Detection was by UV absorption at 260 nm. [Figure 9B] Figure 9B shows chromatograms of the separation of a GalNAc-conjugated oligonucleotide (compound no. 34-01) using an anion-exchange stationary phase (TSK-gel SuperQ-5PW column; 7.5 × 75 mm, 10 μm) with different mobile phases. The oligonucleotide was eluted with an increasing pH gradient from 8.5 to 11 and increasing concentrations of NaBr (trace A) or NaCl (trace B and C). The mobile phase for the separation in trace C had an increasing concentration of acetonitrile compared to the mobile phase for the separation in traces A and B. The mobile phase was applied to the column at a flow rate of 2 mL / min, and the separation was performed at 25 °C. Detection was by UV absorbance at 260 nm. [Figure 9C]Figure 9C shows a preparative chromatogram of the separation of a solution containing GalNAc-conjugated oligonucleotide (compound no. 34-01) using an anion-exchange stationary phase (two TSK-gel SuperQ-5PW columns connected in series; each column: 21.5 × 150 mm, 13 μm) and eluted with a dual pH / salt gradient. The mobile phase consisted of 20 mM NaHPO, 15% acetonitrile (v / v), pH 8.5 (mobile phase A) and 20 mM NaHPO, 15% acetonitrile (v / v), 1 M NaCl, pH 11 (mobile phase B). The gradient conditions were 0–30% mobile phase B from 0–7 min, 30–65% mobile phase B from 7–63 min, 65–70% mobile phase B from 63–63.1 min, and 70% mobile phase B from 63.1–66 min. Re-equilibration with mobile phase A (100%) was performed from 66 to 80 min. The mobile phase was applied to the column at a flow rate of 8.5 mL / min, and separation was performed at ambient temperature. Detection was by UV absorption at 260 nm. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to a preparative purification method for synthetic oligonucleotides, particularly oligonucleotides containing chemically modified nucleotides or modified internucleotide linkages. The method is particularly suitable for separating oligonucleotides conjugated to carbohydrate moieties from unconjugated oligonucleotides and other impurities. In one aspect, the invention is based, in part, on the development of an oligonucleotide purification method using a mixed-mode stationary phase comprising both ion exchange and hydrophobic ligands, and a mobile phase comprising a dual salt / organic solvent gradient that modulates both ion exchange and hydrophobic interactions to control the separation of the target oligonucleotide from impurities. Thus, in certain embodiments, the present invention provides a method for purifying a target oligonucleotide (e.g., a chemically modified oligonucleotide, a carbohydrate-oligonucleotide conjugated compound) from one or more impurities, comprising: contacting a solution containing the target oligonucleotide and one or more impurities with a mixed-mode matrix; passing the mobile phase, which has a pH of about 7.0 to about 8.5 and comprises a buffer, an organic solvent, and an elution salt, through the mixed-mode matrix, the mobile phase containing increasing concentrations of the elution salt and organic solvent over time; and collecting elution fractions from the mixed-mode matrix, wherein one or more impurities are eluted in a first set of elution fractions and the target oligonucleotide is eluted in a second set of elution fractions.

[0021] In another aspect, the present invention relates to methods for purifying oligonucleotides (e.g., carbohydrate-oligonucleotide conjugate compounds) using an anion exchange stationary phase and eluting with a dual pH / salt gradient. Thus, in some embodiments, the present invention provides methods for purifying target oligonucleotides (e.g., chemically modified oligonucleotides, carbohydrate-oligonucleotide conjugate compounds) from one or more impurities, comprising contacting a solution containing the target oligonucleotide and one or more impurities with an anion exchange matrix; passing a mobile phase having a pH of at least about 8.5, comprising a buffer, an organic solvent, and an elution salt, the concentration of the elution salt and the pH of the mobile phase increasing over time, through the anion exchange matrix; and collecting elution fractions from the anion exchange matrix, wherein the target oligonucleotide is eluted in a first set of elution fractions and one or more impurities are eluted in a second set of elution fractions. These methods allow for enhanced selectivity and improved separation between the target oligonucleotide (e.g., carbohydrate-oligonucleotide conjugate compounds) and undesired impurities (e.g., unconjugated oligonucleotides) compared to conventional anion exchange chromatography methods. The improved anion exchange chromatography methods of the present invention can be used in combination with the mixed-mode chromatography methods of the present invention to provide superior purification of oligonucleotides, particularly carbohydrate-oligonucleotide conjugate compounds.

[0022] As used herein, an oligonucleotide refers to an oligomer or polymer of nucleotides. An oligonucleotide may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, or a combination thereof. An oligonucleotide may be several nucleotides to several hundred nucleotides in length, for example, about 10 to about 150 nucleotides, about 12 to about 100 nucleotides, about 15 to about 120 nucleotides, about 20 to about 80 nucleotides, about 10 to about 50 nucleotides, about 14 to about 60 nucleotides, about 15 to about 30 nucleotides, or about 18 to about 26 nucleotides in length. In some embodiments, an oligonucleotide to be purified by the method of the present invention is about 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In one embodiment, the oligonucleotide is about 19 nucleotides in length. In another embodiment, the oligonucleotide is about 20 nucleotides in length. In yet another embodiment, the oligonucleotide is about 21 nucleotides in length. In yet another embodiment, the oligonucleotide is about 23 nucleotides in length.

[0023] The oligonucleotide to be purified according to the method of the present invention can be a natural oligonucleotide isolated from a cell or organism, or it can be a synthetic oligonucleotide produced by chemical synthesis or in vitro enzymatic methods. In some embodiments, the oligonucleotide can be a small hairpin RNA (shRNA), a precursor miRNA (pre-miRNA), an anti-miRNA oligonucleotide (e.g., antagomir and antimiR), or an antisense oligonucleotide. In other embodiments, the oligonucleotide can be one of the component strands of a double-stranded RNA molecule or an RNA interference agent, such as a small interfering RNA (siRNA), a microRNA (miRNA), or an miRNA mimic.

[0024] In certain embodiments, the oligonucleotide to be purified by the methods of the present invention is a therapeutic oligonucleotide designed to target a gene or RNA molecule associated with a disease or disorder. For example, in one embodiment, the oligonucleotide is an antisense oligonucleotide comprising a sequence complementary to a region of the target gene or mRNA sequence. A first sequence is "complementary" to a second sequence if the oligonucleotide comprising the first sequence can hybridize to an oligonucleotide comprising the second sequence under specific conditions to form a double-stranded region. "Hybridize" or "hybridization" refers to the pairing of complementary polynucleotides, typically via hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) between complementary bases in the two oligonucleotides. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the oligonucleotide comprising the first sequence base pairs with the oligonucleotide comprising the second sequence without mismatches across the entire length of one or both nucleotide sequences.

[0025] In some embodiments, the oligonucleotide to be purified by the method of the present invention is the antisense strand of an siRNA or other type of double-stranded RNA interfering agent, and the antisense strand comprises a sequence that is complementary to a region of a target gene or mRNA sequence. In another embodiment, the oligonucleotide is the sense strand of an siRNA or other type of double-stranded RNA interfering agent, and the sense strand comprises a sequence that is identical to a region of a target gene or mRNA sequence. The strand of an siRNA or other type of double-stranded RNA interfering agent that comprises a region having a sequence that is complementary to a target sequence (e.g., target mRNA) is called the "antisense strand." The term "sense strand" refers to the strand that comprises the region that is complementary to the region of the antisense strand.

[0026] The oligonucleotides to be purified according to the methods of the present invention may contain one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide having one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. Such modified nucleotides may include, but are not limited to, nucleotides with 2' sugar modifications (2'-O-methyl, 2'-methoxyethyl, 2'-fluoro, etc.), abasic nucleotides, reverse nucleotides (3'-3' linked nucleotides), phosphorothioate linked nucleotides, nucleotides with bicyclic sugar modifications (e.g., LNA, ENA), and nucleotides containing base analogs (e.g., universal bases, 5-methylcytosine, pseudouracil, etc.).

[0027] In certain embodiments, modified nucleotides have modifications of the ribose sugar. These sugar modifications can include modifications at the 2' and / or 5' positions of the pentose ring as well as bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring with a substituent at the 2' position other than H or OH. Such 2'-modifications include 2'-O-alkyl (e.g., O-C1-C2). 10 Or O-C1~C 10Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. A "bicyclic sugar modification" refers to a modification of the pentose ring in which a bridge connects two atoms of the ring to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides containing sugar moieties with bicyclic sugar modifications are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4'-CH2-O-2') BNAs (also known as locked nucleic acids or LNAs); ethyleneoxy (4'-(CH2)2-O-2') BNAs; aminooxy (4'-CH2-ON(R)-2') BNAs; oxyamino (4'-CH2-N(R)-O-2') BNAs; methyl(methyleneoxy) (4'-CH(CH3)-O-2') BNAs (constrained (c methylene-thio (4'-CH2-S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methyl carbocycle (4'-CH2-CH(CH3)-2') BNA; propylene carbocycle (4'-(CH2)3-2') BNA; and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also called constrained MOE or cMOE).These and other sugar-modified nucleotides that can be incorporated into oligonucleotides to be purified by the methods of the invention are described in U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated herein by reference in their entireties.

[0028] In some embodiments, oligonucleotides to be purified by the methods of the invention comprise one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In particular embodiments, oligonucleotides to be purified by the methods of the invention comprise one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In a particular embodiment, oligonucleotides to be purified by the methods of the invention comprise one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0029] Oligonucleotides that can be purified according to the methods of the present invention can also contain one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage other than a naturally occurring 3' to 5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as a phosphotriester, aminoalkylphosphotriester, alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), phosphinate, phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), phosphorothioate (P=S), phosphorodithioate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate. In one embodiment, the modified internucleotide linkage is a 2' to 5' phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage and, therefore, may be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (—O—Si(H)—O—); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamic acid backbones; methylenemethylimino (—CH—N(CH)—O—CH—) and methylenehydrazino linkages; sulfonic acid and sulfonamide linkages; amide linkages; and others having mixed N, O, S, and CH component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to generate peptide nucleic acids or PNAs, such as those described in U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be incorporated into oligonucleotides to be purified by the methods of the present invention are described in U.S. Pat. Nos. 6,693,187, 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated herein by reference in their entireties.

[0030] In certain embodiments, an oligonucleotide to be purified by the methods of the invention contains one or more phosphorothioate internucleotide linkages. The oligonucleotide may contain 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In some embodiments, all of the internucleotide linkages in the oligonucleotide are phosphorothioate internucleotide linkages. In other embodiments, the oligonucleotide may contain one or more phosphorothioate internucleotide linkages at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus. For example, in certain embodiments, the oligonucleotide contains from about 1 to about 6 or more (e.g., from about 1, 2, 3, 4, 5, 6, or more) consecutive phosphorothioate internucleotide linkages at the 3'-terminus. In other embodiments, the oligonucleotide contains from about 1 to about 6 or more (e.g., from about 1, 2, 3, 4, 5, 6, or more) consecutive phosphorothioate internucleotide linkages at the 5'-terminus. The incorporation of phosphorothioate internucleotide linkages introduces an additional chiral center at the phosphorus of the oligonucleotide, thus generating a diastereomeric pair (Rp and Sp) at each phosphorothioate internucleotide linkage. Diastereomers, or diastereoisomers, are different configurations of compounds that have the same molecular formula and sequence of linked atoms but differ in the three-dimensional orientation of those atoms in space. Unlike enantiomers, diastereomers are not mirror images of each other. Such phosphorothioate diastereomers have the same length, sequence, variance, and mass, making them difficult to separate by most chromatographic approaches. See, for example, Thayer et al., Journal of Chromatography A, Vol. 1218:802-808, 2011. As described in more detail herein, the mixed-mode chromatography method of the present invention provides for the separation of a set of phosphorothioate diastereomers of oligonucleotides on a preparative scale.

[0031] In some embodiments, the oligonucleotide to be purified by the methods of the present invention is conjugated or covalently linked to a ligand that targets the oligonucleotide to a specific tissue or cell type. For example, in one embodiment, the oligonucleotide is covalently linked to a ligand that targets delivery of the oligonucleotide to liver cells (e.g., hepatocytes). One such ligand comprises a carbohydrate that binds to the asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2) expressed on the surface of liver cells. Thus, in certain embodiments, the oligonucleotide to be purified by the methods of the present invention is a carbohydrate-oligonucleotide conjugate compound. A carbohydrate-oligonucleotide conjugate compound refers to an oligonucleotide that is covalently linked to a carbohydrate, either directly or indirectly via a linker moiety. A carbohydrate refers to a compound composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrates incorporated into carbohydrate-oligonucleotide conjugate compounds include monosaccharides selected from pentose, hexose, or heptose, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrates incorporated into carbohydrate-oligonucleotide conjugate compounds include amino sugars such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0032] In some embodiments, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound comprises one or more hexose or hexosamine units. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound comprises one or more glucose, galactose, galactosamine, or glucosamine units. In one embodiment, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound comprises one or more glucose, glucosamine, or N-acetylglucosamine units. In another embodiment, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound comprises one or more galactose, galactosamine, or N-acetyl-galactosamine units. In certain embodiments, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound comprises one or more N-acetyl-galactosamine (GalNAc) units. Examples of GalNAc- or galactose-containing ligands that can be covalently attached to oligonucleotides to produce carbohydrate-oligonucleotide conjugate compounds to be purified according to the methods of the invention are described in U.S. Pat. Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Application Publication Nos. 20030130186 and 20170253875; and WO 2013166155, 2014179620, and 2018039647, all of which are hereby incorporated by reference in their entireties.

[0033] In certain embodiments, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound is a multivalent carbohydrate moiety. As used herein, a multivalent carbohydrate moiety refers to a moiety containing two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety contains two or more binding domains composed of carbohydrates that can bind to two or more different molecules or to two or more different sites on the same molecule. The valency of the carbohydrate moiety indicates the number of individual binding domains within the carbohydrate moiety. For example, the terms monovalent, divalent, trivalent, and tetravalent with respect to a carbohydrate moiety refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A multivalent carbohydrate moiety can include a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound includes a multivalent galactose moiety. In other embodiments, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound includes a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the polyvalent carbohydrate moiety is divalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety can be biantennary or triantennary. In a particular embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Examples of such trivalent and tetravalent galactose- and GalNAc-containing carbohydrate moieties for incorporation into carbohydrate-oligonucleotide conjugate compounds have been previously described. See, e.g., U.S. Pat. Nos. 7,491,805 and 8,106,022; U.S. Patent Application Publication No. 20170253875; and International Publication Nos. 2013166155, 2014179620, and 2018039647. In certain embodiments, the carbohydrate incorporated into the carbohydrate-oligonucleotide conjugate compound comprises a polyvalent N-acetyl-galactosamine moiety having the structure shown in Structure 1 of Example 1.

[0034] In carbohydrate-oligonucleotide conjugate compounds, the carbohydrate can be linked or conjugated to the oligonucleotide directly or indirectly via a linker moiety. The carbohydrate can be linked to the nucleobase, pentose sugar, or internucleotide bond of the oligonucleotide. Conjugation or conjugation to a purine nucleobase or its derivative can occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is linked to a carbohydrate. Conjugation or conjugation to a pyrimidine nucleobase or its derivative can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be linked to a carbohydrate. Conjugation or conjugation to the pentose sugar of a nucleotide can occur at any carbon atom. Representative carbon atoms of a pentose sugar that can be linked to a carbohydrate include the 2', 3', and 5' carbon atoms. The 1' position can also be linked to a carbohydrate, where the nucleobase is removed, such as in an abasic nucleotide. The internucleotide bond can also support a carbohydrate bond. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the carbohydrate can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the carbohydrate can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0035] In certain embodiments, in carbohydrate-oligonucleotide conjugate compounds, the carbohydrate is linked to the 3' or 5' end of the oligonucleotide. In one embodiment, the carbohydrate is covalently linked to the 5' end of the oligonucleotide. In such embodiments, the carbohydrate is linked to the 5'-terminal nucleotide of the oligonucleotide. In these and other embodiments, the carbohydrate is linked to the 5' of the 5'-terminal nucleotide of the oligonucleotide. In other embodiments, the carbohydrate is covalently linked to the 3' end of the oligonucleotide. For example, in some embodiments, the carbohydrate is linked to the 3'-terminal nucleotide of the oligonucleotide. In certain such embodiments, the carbohydrate is linked at the 3' position of the 3'-terminal nucleotide of the oligonucleotide. In alternative embodiments, the carbohydrate is linked near the 3' end of the oligonucleotide but before one or more terminal nucleotides (i.e., before one, two, three, or four terminal nucleotides). In some embodiments, the carbohydrate is linked at the 2' position of the pentose sugar of the 3'-terminal nucleotide of the oligonucleotide. In other embodiments, the carbohydrate is linked at the 2' position of the pentose sugar of the 5'-terminal nucleotide of the oligonucleotide.

[0036] In some carbohydrate-oligonucleotide conjugate compounds to be purified according to the methods of the present invention, the carbohydrate is linked to the oligonucleotide via a linker moiety. The linker moiety is an atom or group of atoms that covalently attaches the carbohydrate to the oligonucleotide. The linker moiety can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker moiety can comprise a bifunctional linking moiety, typically comprising an alkyl moiety bearing two functional groups. One of the functional groups is selected to bind to the oligonucleotide, and the other is selected to bind to some selected group, such as a carbohydrate, essentially as described herein. In certain embodiments, the linker moiety comprises a chain structure or an oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturated (e.g., double or triple bond), and the like. Linker moieties that can be used to link carbohydrates to the present oligonucleotides include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl or substituted or unsubstituted C2-C 10 Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0037] Oligonucleotides to be purified by the methods of the present invention can be readily prepared using techniques known in the art, for example, using conventional solid-phase nucleic acid synthesis. The oligonucleotides can be assembled on a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA). Oligonucleotides can be synthesized via phosphoramidite chemistry using a 2' silyl protecting group at the 5' position of the ribonucleoside in conjunction with acid-labile dimethoxytrityl (DMT). Final deprotection conditions are known not to significantly degrade the RNA product. All syntheses can be performed on large-, medium-, or small-scale scales on any automated or manual synthesizer. Synthesis can also be performed in multiwell plates, columns, or glass slides. The 2'-O-silyl group can be removed via exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluorides. Crown ether catalysts can be utilized in combination with inorganic fluorides in the deprotection reaction. Preferred fluoride ion sources are tetrabutylammonium fluoride or aminohydrofluorides (e.g., combining triethylamine and aqueous HF in a dipolar aprotic solvent, such as dimethylformamide). The various synthetic steps can be performed in an alternative order or sequence to provide the desired compounds.Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on bases) and protecting group techniques (protection and deprotection) useful in the synthesis of oligonucleotides are known in the art, including, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Butts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0038] As can be appreciated by those skilled in the art, further methods for synthesizing the oligonucleotides will be apparent to those skilled in the art. For example, the oligonucleotides can be synthesized in vitro using enzymes, such as those described in Jensen and Davis, Biochemistry, Vol. 57:1821-1832, 2018. Natural oligonucleotides can be isolated from cells or organisms using conventional methods. Custom synthesis of oligonucleotides is also available from several suppliers, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA). Methods for coupling or conjugating carbohydrates to oligonucleotides are also known to those skilled in the art and can include the creation of phosphoramidites of carbohydrate ligands that can be incorporated into standard oligonucleotide synthesis reactions, condensation reactions, ester couplings, and other coupling reactions, the details of which are determined by the type of linker moiety used.

[0039] The methods of the present invention can be used to purify or separate oligonucleotides, particularly carbohydrate-oligonucleotide conjugate compounds, from one or more impurities in a solution. "Purifying" or "purification" refers to a process in which the amount of a substance different from the target molecule (e.g., an oligonucleotide or carbohydrate-oligonucleotide conjugate compound) is reduced and, desirably, eliminated from the final composition or preparation. The term "impurity" refers to a substance having a structure different from the target molecule, and this term can include a single unwanted substance or a combination of several unwanted substances. Impurities can include materials or reagents used in the method for making an oligonucleotide or carbohydrate-oligonucleotide conjugate compound, as well as fragments or other undesired derivatives or forms of the oligonucleotide. In certain embodiments, impurities include one or more oligonucleotides shorter in length than the target oligonucleotide. In these and other embodiments, impurities include one or more failure sequences. Failure sequences can arise during the synthesis of the target oligonucleotide and can result from failed coupling reactions during the stepwise addition of nucleotide monomers to the oligonucleotide chain. The product of an oligonucleotide synthesis reaction is often a heterogeneous mixture of oligonucleotides of various lengths, including the target oligonucleotide and various failure sequences shorter in length than the target oligonucleotide (i.e., truncated versions of the target oligonucleotide). In some embodiments, the impurities include unconjugated oligonucleotides—i.e., oligonucleotides lacking a covalent carbohydrate bond. The presence of unconjugated oligonucleotides may result from an incomplete coupling reaction to link the carbohydrate to the oligonucleotide or from the absence of a carbohydrate component from the conjugated compound as a result of processing or storage conditions. In other embodiments, the impurities include one or more process-related impurities. Depending on the synthetic method used to make the oligonucleotides and / or carbohydrate-oligonucleotide conjugate compounds, such process-related impurities may include, but are not limited to, nucleotide monomers, protecting groups, phosphoramidite precursors, carbohydrate hydrolysis products, salts, enzymes, and endotoxins.

[0040] The solution from which the oligonucleotide or carbohydrate-oligonucleotide conjugate compound can be purified can be any solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more undesirable impurities or contaminants. The solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities can include a mixture resulting from a synthetic method for making the oligonucleotide or conjugate compound. For example, in one embodiment, the solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities is a reaction mixture from a chemical synthesis method for making the oligonucleotide or conjugate compound, such as a synthesis reaction mixture obtained from an automated synthesizer. In such an embodiment, the solution can also contain failure sequences. In another embodiment, the solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities is a mixture from an in vitro enzymatic synthesis reaction (e.g., polymerase chain reaction (PCR)). In yet another embodiment, the solution containing the carbohydrate-oligonucleotide conjugate compound and one or more impurities is a reaction mixture from a coupling reaction for linking a carbohydrate to an oligonucleotide. In yet another embodiment, the solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities is a solution or mixture from another purification operation, such as an eluate from a chromatographic separation. For example, in some embodiments, the solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities is an eluate from an anion exchange chromatography matrix, while in other embodiments, the solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities is an eluate from a mixed-mode chromatography matrix.

[0041] In certain embodiments, the present invention provides methods for purifying oligonucleotides, particularly carbohydrate-oligonucleotide conjugate compounds, from one or more impurities by contacting a solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities with a mixed-mode matrix and eluting the oligonucleotide or carbohydrate-oligonucleotide conjugate compound from the mixed-mode matrix using a mobile phase comprising a buffer and increasing concentrations of an elution salt and an organic solvent (e.g., a dual salt / organic solvent gradient). In one embodiment, the method includes contacting a solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities with the mixed-mode matrix; passing a mobile phase having a pH of about 7.0 to about 8.5, comprising a buffer, an organic solvent, and an elution salt, the concentration of the elution salt and organic solvent increasing over time, through the mixed-mode matrix; collecting elution fractions from the mixed-mode matrix, wherein the one or more impurities are eluted in a first set of elution fractions and the oligonucleotide or carbohydrate-oligonucleotide conjugate compound is eluted in a second set of elution fractions, thereby separating the oligonucleotide or carbohydrate-oligonucleotide conjugate compound from the impurities.

[0042] Thus, in some embodiments, the methods of the present invention involve contacting an oligonucleotide or carbohydrate-oligonucleotide conjugate compound with a mixed-mode matrix. A mixed-mode matrix refers to a material containing ligands with functional groups that interact with solutes through two or more modes or mechanisms of interaction. For example, a mixed-mode matrix may contain ligands with one set of functional groups that interact with solutes based on charge-charge interactions and a second set of functional groups that interact with solutes based on hydrophobic or hydrophilic interactions. Mixed-mode matrices can be created by a variety of approaches, including, but not limited to, (i) combining two or more types of particles, each with ligands having different functional groups (e.g., ion-exchange ligands and hydrophobic ligands), into a single column; (ii) immobilizing different sets of ligands with different functional groups on a support; and (iii) tethering two or more ligands with different functional groups to a support. The support to which the ligands with different functional groups are tethered is typically composed of silica gel or cross-linked polymers, such as polymethacrylate, polyvinylpyrrolidone-divinylbenzene, and polystyrene-divinylbenzene, although other materials can also be used.

[0043] In some embodiments, the pore size of the mixed mode matrix is ​​less than about 20 nm. For example, the pore size of the mixed mode matrix can be about 5 nm to about 18 nm, about 8 nm to about 15 nm, about 10 nm to about 16 nm, about 7 nm to about 14 nm, or about 11 nm to about 14 nm. In specific embodiments, the mixed mode matrix has a pore size of about 8 nm to about 15 nm. In some embodiments, the mixed mode matrix has a pore size of about 11 nm to about 14 nm. In one specific embodiment, the mixed mode matrix has a pore size of about 13 nm. In another specific embodiment, the mixed mode matrix has a pore size of about 10 nm.

[0044] The mixed-mode matrices used in the methods of the present invention generally contain ligands having positively charged functional groups, negatively charged functional groups, and hydrophobic functional groups. The positively charged functional groups can be primary amines, secondary amines, tertiary amines, or quaternary amines. The negatively charged functional groups can be sulfonyl groups (e.g., sulfoethyl, sulfopropyl, sulfonate), carboxyl groups (e.g., carboxymethyl, carboxylate), or phosphate groups (e.g., phosphonate). The hydrophobic functional groups can be alkyl groups (e.g., isopropyl, propyl, t-butyl, butyl, and C8-C18 alkyl chains) or aryl groups (e.g., phenyl).

[0045] In certain embodiments, the mixed-mode matrix contains strong ion-exchange ligands, which refers to ligands containing strong ion-exchange groups. Strong anion-exchange groups do not exhibit a change in ion-exchange capacity with changes in pH and are fully charged within a wide pH range (e.g., pH values ​​from 2 to 13). Strong anion-exchange ligands can include quaternary amines, such as quaternary aminoethyl, quaternary ammonium, and quaternary aminomethyl. Strong cation-exchange ligands can include sulfonyl functional groups, such as sulfoethyl, sulfopropyl, and sulfonate.

[0046] In another embodiment, the mixed-mode matrix contains weak ion-exchange ligands, which refers to ligands containing weak ion-exchange groups. Weak anion-exchange groups are ionized only in a limited pH range. Examples of weak anion-exchange groups include, but are not limited to, polyethyleneimine, diethylaminomethyl, diethylaminoethyl, dimethylaminopropyl, ethylenediamino, and polyallylamine. Representative weak cation-exchange groups include, but are not limited to, phosphate groups, such as phosphonates, and carboxyl groups, such as carboxymethyl and carboxylate.

[0047] In some embodiments, the mixed-mode matrix comprises a high density of ion-exchange ligands, particularly strong ion-exchange ligands, such that the mixed-mode matrix has a high ion-exchange capacity. For example, the mixed-mode matrix can have a density of strong ion-exchange ligands greater than about 100 μmol / gram, greater than about 150 μmol / gram, greater than about 200 μmol / gram, greater than about 250 μmol / gram, greater than about 300 μmol / gram, greater than about 350 μmol / gram, greater than about 400 μmol / gram, or greater than about 450 μmol / gram.

[0048] Ion exchange capacity may be expressed as microequivalents (μeq) / mL matrix. In certain embodiments, the mixed-mode matrix has an anion exchange capacity of at least 4 μeq / mL, at least 5 μeq / mL, at least 6 μeq / mL, at least 7 μeq / mL, or at least 8 μeq / mL matrix. In some embodiments, the mixed-mode matrix has an anion exchange capacity of about 6 μeq / mL to about 10 μeq / mL matrix. In other embodiments, the mixed-mode matrix has an anion exchange capacity of about 7 μeq / mL to about 9 μeq / mL matrix. In these and other embodiments, the mixed-mode matrix has a cation exchange capacity of at least 8 μeq / mL, at least 10 μeq / mL, at least 12 μeq / mL, at least 14 μeq / mL, at least 16 μeq / mL, at least 18 μeq / mL, or at least 20 μeq / mL matrix. In some embodiments, the mixed-mode matrix has a cation exchange capacity of about 14 μeq / mL to about 24 μeq / mL matrix. In other embodiments, the mixed-mode matrix has a cation exchange capacity of about 18 μeq / mL to about 22 μeq / mL matrix. In one particular embodiment, the mixed-mode matrix has an anion exchange capacity of about 7 μeq / mL to about 9 μeq / mL matrix and a cation exchange capacity of about 18 μeq / mL to about 22 μeq / mL matrix. The ion exchange capacities of various matrices can be measured according to methods known to those skilled in the art, such as those described in Kazarian et al., Anal Chim Acta, Vol. 803:143-153, 2013 and Kazarian et al., Chromatographia, Vol. 78:179-187, 2015.

[0049] In addition to ion exchange ligands, the mixed-mode matrix used in the methods of the present invention generally also includes hydrophobic ligands, which are ligands containing hydrophobic functional groups. The hydrophobic ligands can include alkyl groups, such as propyl, butyl, isopropyl, t-butyl, or longer alkyl chains (e.g., C8-C18 alkyl chains), aryl groups, such as phenyl groups, or mixtures thereof. In one embodiment, the mixed-mode matrix includes hydrophobic ligands containing alkyl groups, such as C8 or C18 alkyl chains. In another embodiment, the mixed-mode matrix includes hydrophobic ligands containing phenyl groups.

[0050] In certain preferred embodiments, the mixed-mode matrix used in the methods of the present invention comprises strong anion-exchange ligands, strong cation-exchange ligands, and hydrophobic ligands. In one embodiment, the strong anion-exchange ligand comprises a quaternary amine, the strong cation-exchange ligand comprises a sulfonyl functional group, and the hydrophobic ligand comprises an alkyl group. In such embodiments, the alkyl group is an alkyl chain comprising at least 8 carbon atoms. For example, in one embodiment, the alkyl group comprises an octyl carbon chain (C alkyl chain). In another embodiment, the alkyl group comprises an octadecyl carbon chain (C alkyl chain). In other embodiments, the strong anion-exchange ligand comprises a quaternary amine, the strong cation-exchange ligand comprises a sulfonyl functional group, and the hydrophobic ligand comprises a phenyl group.

[0051] Suitable mixed-mode matrices for use in the methods of the invention are also described in Zhang and Liu, Journal of Pharmaceutical and Biomedical Analysis, Vol. 128:73-88, 2016, and are also commercially available, such as the Scherzo line of columns available from Imtakt USA, including SW-C18, SM-C18, and SS-C18 columns. The Scherzo SS-C18 column is preferred in some embodiments as a mixed-mode matrix for use in the methods of the invention.

[0052] Once a solution containing oligonucleotides or carbohydrate-oligonucleotide conjugate compounds and one or more impurities is contacted with the mixed-mode matrix, a mobile phase is passed through the mixed-mode matrix, carrying the solution components through the matrix and allowing the components to interact to varying degrees with the positively charged, negatively charged, and hydrophobic functional groups present in the matrix. As described in Example 1 herein, the composition of the mobile phase was designed to take advantage of both the ion exchange and reversed-phase interactions of the mixed-mode matrix to improve the separation of carbohydrate-oligonucleotide conjugate compounds from unconjugated oligonucleotides and other impurities.

[0053] The mobile phase used in the mixed-mode chromatography methods of the present invention is generally a buffer solution with a pH of about 7.0 to about 8.5. In some embodiments, the mobile phase has a pH of about 7.0 to about 8.0. In other embodiments, the mobile phase has a pH of about 7.3 to about 7.7. In one particular embodiment, the mobile phase has a pH of about 7.5. Any buffer may be used, provided that the buffer is capable of maintaining the pH of the solution in the target pH range. Suitable buffers that provide buffering in this pH range and can be used as components of the mobile phase in the mixed-mode chromatography methods of the present invention include HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Tris hydrochloride, phosphate, BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tricine (N-Tris[hydroxymethyl]methylglycine), Bicine (N,N-bis(2-hydroxyethyl)glycine), and the like. Examples of suitable mobile phase buffers include, but are not limited to, TES (N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), bis-tris (bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane), and MOPS (3-[N-morpholino]propanesulfonic acid). In certain embodiments, the mobile phase comprises a buffer selected from sodium phosphate, Tris hydrochloride, HEPES, or MOPS. The buffer may be present at a concentration of about 20 mM to about 200 mM, about 25 mM to about 175 mM, about 40 mM to about 150 mM, about 50 mM to about 125 mM, or about 80 mM to about 110 mM. In some embodiments, the mobile phase comprises a Tris-hydrochloride buffer, e.g., at a concentration of about 20 mM to about 200 mM. In other embodiments, the mobile phase comprises a HEPES buffer, e.g., at a concentration of about 20 mM to about 200 mM. In certain other embodiments, the mobile phase comprises a sodium phosphate buffer, e.g., at a concentration of about 20 mM to about 200 mM. In still other embodiments, the mobile phase comprises a MOPS buffer, e.g., at a concentration of about 20 mM to about 200 mM.

[0054] In certain embodiments, the mobile phase used in the mixed-mode chromatography method of the present invention comprises an organic solvent. Exemplary organic solvents that may be included in the mobile phase include, but are not limited to, acetonitrile, methanol, propanol, isopropanol, ethanol, butanol, tetrahydrofuran, and acetone. In some embodiments, the organic solvent is methanol, acetonitrile, or tetrahydrofuran. In one embodiment, the organic solvent is acetonitrile. In another embodiment, the organic solvent is methanol. In certain embodiments, the concentration of the organic solvent in the mobile phase is increased over the course of the separation. As described in Example 1, increasing the concentration of organic solvent (e.g., acetonitrile) in the mobile phase utilized the reversed-phase mode of a mixed-mode matrix to enable elution of oligonucleotides from the matrix.

[0055] In some embodiments, the concentration of organic solvent in the mobile phase at the start of a separation is at least about 8% (v / v) and increases over the course of the separation. In other embodiments, the concentration of organic solvent in the mobile phase at the start of a separation is at least about 10% (v / v) and increases over the course of the separation. In certain embodiments, the increasing concentration of organic solvent in the mobile phase is, for example, a gradient of organic solvent from about 8% (v / v) to about 20% (v / v), from about 10% (v / v) to about 18% (v / v), from about 10% (v / v) to about 20% (v / v), from about 8% (v / v) to about 14% (v / v), from about 9% (v / v) to about 16% (v / v), or from about 11% (v / v) to about 17% (v / v). In one embodiment, the concentration of organic solvent in the mobile phase increases from about 8% (v / v) to about 20% (v / v) over the course of the separation. In another embodiment, the concentration of organic solvent in the mobile phase increases from about 10% (v / v) to about 18% (v / v) over the series of separations, hi yet another embodiment, the concentration of organic solvent in the mobile phase increases from about 11% (v / v) to about 17% (v / v) over the series of separations.

[0056] The organic solvent gradient can be a linear gradient, in which the concentration of the organic solvent in the mobile phase changes linearly over time. In other embodiments, the organic solvent gradient is a step gradient, in which the concentration of the organic solvent in the mobile phase changes over time in discrete steps, with the organic solvent concentration at each step being constant. Both types of gradients can be generated by mixing two buffers with different percentages of organic solvent at different times. As an example, to generate a gradient, Buffer A containing no organic solvent can be mixed with Buffer B containing 20% ​​(v / v) organic solvent. Increasing the percentage of Buffer B in the mixture with Buffer A as a function of time allows for the generation of a linear concentration gradient of organic solvent from 0% to 20% (v / v). Similarly, step gradients can be generated by mixing specified percentages of Buffer A and Buffer B at specific points during a separation, as illustrated in the Examples and described in more detail below.

[0057] In some embodiments, the mobile phase used in the mixed-mode chromatography method of the present invention contains an eluting salt, the concentration of which increases over the course of the separation. The eluting salt refers to an ionic compound resulting from the neutralization reaction of an acid and a base. The salt typically consists of equal numbers of cations and anions such that the overall net charge of the salt is zero. Suitable cations in the eluting salt include, but are not limited to, sodium, potassium, ammonium, trimethylammonium, triethylammonium, lithium, calcium, and magnesium. In certain embodiments, the cation in the eluting salt may be selected from sodium, potassium, ammonium, trimethylammonium, and triethylammonium. In some embodiments, the cation in the eluting salt is sodium, potassium, or ammonium. In one embodiment, the cation in the eluting salt is sodium. In another embodiment, the cation in the eluting salt is potassium. Suitable anions in the eluting salt include, but are not limited to, chloride, bromide, nitrate, nitrite, iodide, perchlorate, acetate, formate, phosphate, citrate, oxalate, and carbonate. The anion in the eluting salt may be chloride, bromide, nitrate, nitrite, iodide, perchlorate, acetate, or formate in some embodiments. In one particular embodiment, the anion in the eluting salt is chloride. In another particular embodiment, the anion in the eluting salt is bromide.

[0058] Exemplary eluting salts that may be included in the mobile phase for the mixed-mode chromatography method of the present invention include, but are not limited to, sodium chloride, sodium bromide, sodium nitrate, sodium nitrite, sodium acetate, sodium perchlorate, sodium iodide, sodium formate, potassium chloride, potassium bromide, potassium nitrate, potassium nitrite, potassium acetate, potassium perchlorate, potassium iodide, potassium formate, ammonium chloride, ammonium bromide, ammonium acetate, trimethylammonium chloride, trimethylammonium bromide, trimethylammonium acetate, triethylammonium chloride, triethylammonium bromide, and triethylammonium acetate. In certain embodiments, the mobile phase comprises an eluting salt selected from sodium bromide, potassium bromide, ammonium bromide, sodium chloride, potassium chloride, and ammonium chloride. In one embodiment, the eluting salt in the mobile phase is sodium bromide. In another embodiment, the eluting salt in the mobile phase is potassium bromide. In another embodiment, the eluting salt in the mobile phase is ammonium bromide. In yet another embodiment, the eluting salt in the mobile phase is sodium chloride.

[0059] During separation, the concentration of eluting salt in the mobile phase is increased to disrupt the electrostatic interactions between the oligonucleotides and carbohydrate-oligonucleotide conjugate compounds and the ion-exchange ligands in the mixed-mode matrix. The increasing concentration of eluting salt in the mobile phase can be a concentration gradient of eluting salt, e.g., from 0 M to about 2 M, from 0 M to about 1 M, from 0 M to about 0.5 M, from about 0.5 M to about 1 M, or from about 0.5 M to about 2 M. In some embodiments, the gradient is a linear gradient, in which the concentration of eluting salt in the mobile phase changes linearly over time. In other embodiments, the gradient is a step gradient, in which the concentration of eluting salt in the mobile phase changes over time in discrete steps, with the eluting salt concentration at each step being constant. As described above for generating organic solvent concentration gradients, both linear and step gradients of eluting salt can be similarly generated by mixing different percentages of two buffers with different concentrations of eluting salt for different times. In one embodiment, the increasing concentration of eluting salt in the mobile phase is a linear gradient from about 0.5 M to about 1 M. In another embodiment, the increasing elution salt concentration in the mobile phase is a linear gradient from about 0.5 M to about 0.85 M. In yet another embodiment, the increasing elution salt concentration in the mobile phase is a step gradient from about 0.5 M to about 1 M. In yet another embodiment, the increasing elution salt concentration in the mobile phase is a step gradient from about 0.5 M to about 0.85 M.

[0060] In certain preferred embodiments, the mobile phase used in the mixed-mode chromatography methods of the present invention comprises a dual salt / organic solvent gradient. Accordingly, in some embodiments, the concentrations of organic solvent and elution salt in the mobile phase are increased over the course of the separation. In one embodiment, the mobile phase comprises an organic solvent gradient of about 8% (v / v) to about 20% (v / v) and an elution salt gradient of 0 M to about 1 M. In another embodiment, the mobile phase comprises an organic solvent gradient of about 8% (v / v) to about 20% (v / v) and an elution salt gradient of about 0.5 M to about 1 M. In yet another embodiment, the mobile phase comprises an organic solvent gradient of about 10% (v / v) to about 18% (v / v) and an elution salt gradient of about 0.5 M to about 1 M. In yet another embodiment, the mobile phase comprises an organic solvent gradient of about 11% (v / v) to about 17% (v / v) and an elution salt gradient of about 0.5 M to about 0.85 M. A representative dual salt / organic solvent gradient for the mobile phase for the mixed-mode chromatography method of the present invention is set forth in the following table, where Buffer A contains no eluting salt (i.e., 0 M) or no organic solvent (i.e., 0% (v / v)), and Buffer B contains 1 M eluting salt and 20% (v / v) organic solvent.

[0061] [Table 1]

[0062] Other possible gradients and methods for generating gradients for increasing the concentration of eluting salts and / or organic solvents in the mobile phase over a series of separations will be known to those skilled in the art.

[0063] In some embodiments of the mixed-mode chromatography method of the present invention, the mobile phase has a pH of about 7.0 to about 8.5 and contains about 20 mM to about 200 mM of buffer, organic solvent, and elution salt, with the organic solvent concentration increasing at a gradient of about 8% (v / v) to about 20% (v / v) over time, and the elution salt concentration increasing at a gradient of 0 M to about 1 M. In other embodiments, the mobile phase has a pH of about 7.0 to about 8.0 and contains about 20 mM to about 200 mM of buffer, organic solvent, and elution salt, with the organic solvent concentration increasing at a gradient of about 10% (v / v) to about 18% (v / v) over time, and the elution salt concentration increasing at a gradient of about 0.5 M to about 1 M. For any of these mobile phase compositions, the buffer can be sodium phosphate or Tris hydrochloride, the organic solvent can be acetonitrile or methanol, and the elution salt can be sodium bromide, potassium bromide, or ammonium bromide. For example, in certain embodiments, the mobile phase has a pH of about 7.0 to about 8.5 and contains about 20 mM to about 200 mM Tris hydrochloride buffer, acetonitrile, and sodium bromide, and the acetonitrile concentration in the mobile phase is increased over time at a gradient of about 8% (v / v) to about 20% (v / v) and the sodium bromide concentration in the mobile phase is increased over time at a gradient of 0 M to about 1 M. In some embodiments, the mobile phase has a pH of about 7.0 to about 8.0 and contains about 20 mM to about 200 mM Tris hydrochloride buffer, acetonitrile, and sodium bromide, and the acetonitrile concentration in the mobile phase is increased over time at a gradient of about 8% (v / v) to about 20% (v / v) and the sodium bromide concentration in the mobile phase is increased over time at a gradient of about 0.5 M to about 1 M. In another embodiment, the mobile phase has a pH of about 7.3 to about 7.7 and comprises about 80 mM to about 110 mM Tris hydrochloride buffer, acetonitrile, and sodium bromide, wherein the acetonitrile concentration in the mobile phase is increased over time at a gradient of about 10% (v / v) to about 18% (v / v), and the sodium bromide concentration in the mobile phase is increased over time at a gradient of about 0.5 M to about 1 M. In one embodiment, the mobile phase has a pH of about 7.5 and comprises about 100 mM Tris hydrochloride buffer, acetonitrile, and sodium bromide, wherein the acetonitrile concentration in the mobile phase is increased over time at a gradient of about 11% (v / v) to about 17% (v / v), and the sodium bromide concentration in the mobile phase is increased over time at a gradient of about 0.5 M to about 0.85 M.

[0064] In certain embodiments of the mixed-mode chromatography method of the present invention, the mobile phase has a pH of about 7.0 to about 8.5 and comprises about 20 mM to about 200 mM Tris-hydrochloride buffer, methanol, and sodium bromide, and the methanol concentration in the mobile phase is increased over time at a gradient of about 8% (v / v) to about 20% (v / v) and the sodium bromide concentration in the mobile phase is increased over time at a gradient of 0 M to about 1 M. In some embodiments, the mobile phase has a pH of about 7.0 to about 8.0 and comprises about 20 mM to about 200 mM Tris-hydrochloride buffer, methanol, and sodium bromide, and the methanol concentration in the mobile phase is increased over time at a gradient of about 8% (v / v) to about 20% (v / v) and the sodium bromide concentration in the mobile phase is increased over time at a gradient of about 0.5 M to about 1 M. In another embodiment, the mobile phase has a pH of about 7.3 to about 7.7 and contains about 80 mM to about 110 mM Tris hydrochloride buffer, methanol, and sodium bromide, and the methanol concentration in the mobile phase is increased over time at a gradient of about 10% (v / v) to about 18% (v / v) and the sodium bromide concentration in the mobile phase is increased over time at a gradient of about 0.5 M to about 1 M. In yet another embodiment, the mobile phase has a pH of about 7.0 to about 8.5 and contains about 20 mM to about 200 mM sodium phosphate buffer, methanol, and sodium bromide, and the methanol concentration in the mobile phase is increased over time at a gradient of about 8% (v / v) to about 20% (v / v) and the sodium bromide concentration in the mobile phase is increased over time at a gradient of 0 M to about 1 M. In still other embodiments, the mobile phase has a pH of about 7.0 to about 8.0 and comprises about 20 mM to about 200 mM sodium phosphate buffer, methanol, and sodium bromide, the methanol concentration in the mobile phase increasing over time at a gradient of about 8% (v / v) to about 20% (v / v) and the sodium bromide concentration in the mobile phase increasing over time at a gradient of about 0.5 M to about 1 M. In certain other embodiments, the mobile phase has a pH of about 7.0 to about 8.5 and comprises about 20 mM to about 200 mM sodium phosphate buffer, acetonitrile, and sodium bromide, the acetonitrile concentration in the mobile phase increasing over time at a gradient of about 8% (v / v) to about 20% (v / v) and the sodium bromide concentration in the mobile phase increasing over time at a gradient of 0 M to about 1 M.In yet another embodiment, the mobile phase has a pH of about 7.0 to about 8.0 and contains about 20 mM to about 200 mM sodium phosphate buffer, acetonitrile, and sodium bromide, and the acetonitrile concentration in the mobile phase is increased over time at a gradient of about 8% (v / v) to about 20% (v / v), and the sodium bromide concentration in the mobile phase is increased over time at a gradient of about 0.5 M to about 1 M. In any of the above mobile phase buffers, potassium bromide or ammonium bromide can be used as the eluting salt instead of sodium bromide.

[0065] As the solution containing the oligonucleotide / carbohydrate-oligonucleotide conjugate compound and one or more impurities moves through the mixed-mode matrix with the mobile phase, elution fractions are collected. UV absorption, e.g., at 260 nm, can be used to monitor the oligonucleotide content of the fractions. As shown by the chromatogram in Figure 2, when mixed-mode chromatography is operated according to the methods of the present invention, unconjugated oligonucleotides (e.g., impurities in this content) are eluted from the mixed-mode matrix before the carbohydrate-oligonucleotide conjugate compound (e.g., GalNAc-oligo), thus allowing the collection of a set of fractions for the carbohydrate-oligonucleotide conjugate compound separately from one or more impurities. To verify the enrichment of fractions for carbohydrate-oligonucleotide conjugate compounds or other target oligonucleotides, samples from the elution fractions can be analyzed by gel electrophoresis, capillary electrophoresis, ion-pairing reversed-phase liquid chromatography-mass spectrometry, analytical ion-exchange chromatography, and / or native mass spectrometry.

[0066] In certain embodiments, a target oligonucleotide or carbohydrate-oligonucleotide conjugate compound to be purified by the mixed-mode chromatography method of the present invention contains one or more phosphorothioate internucleotide linkages. As discussed above, the incorporation of phosphorothioate internucleotide linkages generates a diastereomeric pair (Rp and Sp) at each such linkage. As described in Example 2 herein, the mixed-mode chromatography method of the present invention enables the separation of a set of phosphorothioate diastereomers. Thus, in some embodiments, the method includes contacting a solution containing an oligonucleotide containing at least one phosphorothioate internucleotide linkage and one or more phosphorothioate diastereomers of the oligonucleotide with a mixed-mode matrix as described herein; passing a mobile phase having a pH of about 7.0 to about 8.5, comprising a buffer, an organic solvent, and an elution salt, the concentration of the elution salt and organic solvent increasing over time, through the mixed-mode matrix; and collecting elution fractions from the mixed-mode matrix, wherein a first diastereomer of the oligonucleotide is eluted in a separate set of elution fractions, separate from a second diastereomer of the oligonucleotide. In certain embodiments, the method includes contacting a solution containing a carbohydrate-oligonucleotide conjugate compound containing at least one phosphorothioate internucleotide linkage and one or more phosphorothioate diastereomers of the conjugated compound with a mixed-mode matrix as described herein; passing a mobile phase having a pH of about 7.0 to about 8.5, the mobile phase comprising a buffer, an organic solvent, and an elution salt, the concentration of the elution salt and organic solvent increasing over time, through the mixed-mode matrix; collecting elution fractions from the mixed-mode matrix, wherein a first diastereomer of the conjugated compound is eluted in a separate set of elution fractions, separate from a second diastereomer of the conjugated compound. In some embodiments, the method can further include isolating the set of elution fractions containing a particular phosphorothioate diastereomer or the set of phosphorothioate diastereomers.

[0067] In another aspect, the present invention provides a method for purifying oligonucleotides, particularly carbohydrate-oligonucleotide conjugate compounds, from one or more impurities by contacting a solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities with an anion exchange matrix and eluting the oligonucleotide or carbohydrate-oligonucleotide conjugate compound from the anion exchange matrix using a dual pH / salt gradient. In one embodiment, the method includes contacting a solution containing the oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities with an anion exchange matrix; passing a mobile phase having a pH of at least about 8.5 and comprising a buffer, an organic solvent, and an elution salt, through the anion exchange matrix, wherein the concentration of the elution salt and the pH of the mobile phase increase over time; collecting elution fractions from the anion exchange matrix, wherein the oligonucleotide or carbohydrate-oligonucleotide conjugate compound is eluted in a first set of elution fractions and the one or more impurities are eluted in a second set of elution fractions, thereby separating the oligonucleotide or carbohydrate-oligonucleotide conjugate compound from the impurities.

[0068] Thus, in this embodiment of the method of the present invention, a solution containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound to be purified is contacted with an anion exchange matrix. An anion exchange matrix refers to a material to which one or more anion exchange ligands are attached. Anion exchange ligands generally contain positively charged or positively charged functional groups. The material to which the anion exchange ligands are attached can be made from polymers such as cross-linked carbohydrates including agarose, agar, cellulose, dextran, and chitosan, or cross-linked synthetic polymers including styrene or styrene derivatives, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinylamides, and the like. Other suitable materials include inorganic polymers such as silica. The material can be in the form of beads or particles.

[0069] In certain embodiments, the anion exchange matrix comprises a strong anion exchange ligand, which refers to a ligand containing a strong anion exchange group that shows no change in ion exchange capacity with pH and is fully charged over a wide pH range (e.g., pH values ​​from 2 to 13). Strong anion exchange ligands can include quaternary amines, such as quaternary aminoethyl, quaternary ammonium, and quaternary aminomethyl. Anion exchange matrices suitable for use in the methods of the present invention are also commercially available, such as the TSKgel SuperQ-5PW column available from Tosoh Bioscience, the Source Q15 and Q30 columns available from GE Healthcare, and the DNAPac PA100 and PA200 columns available from ThermoFisher Scientific. In some embodiments, the TSKgel SuperQ-5PW column is preferred as an anion exchange matrix for use in the methods of the present invention. Other strong anion exchange matrices can also be used in the methods of the present invention, as long as the matrix is ​​stable over the pH range (e.g., pH 8.5 to 12) used for the mobile phase pH gradient.

[0070] The mobile phase for eluting oligonucleotides or carbohydrate-oligonucleotide conjugate compounds from an anion exchange matrix typically comprises a buffer, an organic solvent, and an elution salt. In some embodiments, the mobile phase initially has a pH of at least about 8.5, and the pH is increased over the course of the separation. As described in Example 3 herein, the pH change allows for tuning the ionization of the carbohydrate-oligonucleotide conjugate compound without affecting the ionization of the strong anion exchange matrix to improve separation of the conjugated compound from unconjugated oligonucleotides and other impurities. Thus, in certain embodiments, the pH of the mobile phase is increased over the course of the separation from about 8.5 to about 12, from about 8.5 to about 11, from about 8.5 to about 10.5, from about 8.5 to about 9.5, from about 9.0 to about 10.5, from about 9.5 to about 10.5, or from about 9.5 to about 11. In some embodiments, the pH of the mobile phase is increased over the course of the separation from about 8.5 to about 11. In other embodiments, the pH of the mobile phase is increased over the course of the separation from about 9.0 to about 10.5. In yet another embodiment, the pH of the mobile phase is increased from about 8.5 to about 10.5 over the course of the separation. Any buffer can be used, provided that it is capable of maintaining the pH of the mobile phase over the range of the pH gradient. Suitable buffers that buffer over the target pH gradient range and can be used as components of the mobile phase in the ion exchange chromatography methods of the present invention include, but are not limited to, phosphate, glycine, carbonate, bicarbonate, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CABS (4-(cyclohexylamino)-1-butanesulfonic acid), CHES (2-(cyclohexylamino)ethanesulfonic acid), AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), AMP (2-amino-2-methyl-1-propanol), and AMPD (2-amino-2-methyl-1,3-propanediol). In one embodiment, the mobile phase comprises a sodium phosphate buffer, which may be present at a concentration of about 10 mM to about 200 mM, about 15 mM to about 150 mM, about 20 mM to about 100 mM, about 25 mM to about 75 mM, or about 15 mM to about 25 mM.In some embodiments, the mobile phase comprises a sodium phosphate buffer, for example, at a concentration of about 20 mM to about 100 mM.

[0071] In certain embodiments, the mobile phase used in the anion exchange chromatography method of the present invention comprises an organic solvent. Exemplary organic solvents that may be included in the mobile phase include, but are not limited to, acetonitrile, methanol, propanol, isopropanol, ethanol, butanol, tetrahydrofuran, and acetone. In some embodiments, the organic solvent is methanol, acetonitrile, or tetrahydrofuran. In one embodiment, the organic solvent is acetonitrile. In another embodiment, the organic solvent is methanol. The concentration of the organic solvent in the mobile phase used in the anion exchange chromatography method of the present invention can be about 1% (v / v) to about 50% (v / v), about 1% (v / v) to about 20% (v / v), about 15% (v / v) to about 35% (v / v), about 5% (v / v) to about 15% (v / v), about 5% (v / v) to about 25% (v / v), about 1% (v / v) to about 10% (v / v), about 10% (v / v) to about 20% (v / v), about 8% (v / v) to about 12% (v / v), or about 12% (v / v) to about 18% (v / v). In one embodiment, the organic solvent is present in the mobile phase at a concentration of about 10% (v / v). In another embodiment, the organic solvent is present in the mobile phase at a concentration of about 15% (v / v). In another embodiment, the organic solvent is present in the mobile phase at a concentration of about 20% (v / v). In certain embodiments, the concentration of organic solvent in the mobile phase used in the anion exchange chromatography method of the invention remains constant throughout the separation.

[0072] In some embodiments, the mobile phase used in the anion exchange chromatography method of the present invention comprises an eluting salt, the concentration of which increases over the course of the separation. Any of the eluting salts described above for use in the mobile phase for the mixed-mode chromatography method of the present invention can also be used in the mobile phase for the anion exchange chromatography method. For example, suitable cations in the eluting salt may include, but are not limited to, sodium, potassium, ammonium, trimethylammonium, triethylammonium, lithium, calcium, and magnesium. In certain embodiments, the cation in the eluting salt may be selected from sodium, potassium, ammonium, trimethylammonium, and triethylammonium. In some embodiments, the cation in the eluting salt is sodium, potassium, or ammonium. In one embodiment, the cation in the eluting salt is sodium. In another embodiment, the cation in the eluting salt is potassium. Suitable anions in the eluting salt may include, but are not limited to, chloride, bromide, nitrate, nitrite, iodide, perchlorate, acetate, formate, phosphate, citrate, oxalate, and carbonate. In some embodiments, the anion in the eluting salt is chloride, bromide, nitrate, nitrite, iodine, perchlorate, acetate, or formate. In a particular embodiment, the anion in the eluting salt is chloride. In another particular embodiment, the anion in the eluting salt is bromide. Representative eluting salts that can be included in the mobile phase for the anion effect chromatography method of the present invention include, but are not limited to, sodium chloride, sodium bromide, sodium nitrite, sodium acetate, sodium perchlorate, sodium iodide, sodium formate, potassium chloride, potassium bromide, potassium nitrate, potassium nitrite, potassium acetate, potassium perchlorate, potassium iodide, potassium formate, ammonium chloride, ammonium bromide, ammonium acetate, trimethylammonium chloride, trimethylammonium bromide, trimethylammonium acetate, triethylammonium chloride, triethylammonium bromide, and triethylammonium acetate. In certain embodiments, the mobile phase comprises an eluting salt selected from sodium bromide, potassium bromide, ammonium bromide, sodium chloride, potassium chloride, and ammonium chloride.In one embodiment, the eluting salt in the mobile phase for the anion exchange chromatography method of the present invention is sodium chloride. In another embodiment, the eluting salt in the mobile phase is sodium bromide. In another embodiment, the eluting salt in the mobile phase is potassium chloride. In yet another embodiment, the eluting salt in the mobile phase is ammonium chloride.

[0073] During separation, the concentration of eluting salt in the mobile phase is increased to disrupt the electrostatic interactions between the oligonucleotides and carbohydrate-oligonucleotide conjugate compounds and the positively charged functional groups in the anion exchange matrix. For an anion exchange chromatography method of the present invention, the increasing concentration of eluting salt in the mobile phase can be, for example, a concentration gradient of eluting salt from 0 M to about 2 M, 0 M to about 1 M, 0 M to about 0.5 M, about 0.5 M to about 1 M, about 0.3 M to about 0.7 M, about 0.2 M to about 0.8 M, or about 0.5 M to about 2 M. In some embodiments, the gradient is a linear gradient, in which the concentration of eluting salt in the mobile phase changes linearly over time. In other embodiments, the gradient is a stepwise gradient, in which the concentration of eluting salt in the mobile phase changes over time in discrete steps, with the eluting salt concentration at each step remaining constant. As described above for the elution salt gradient in the mobile phase buffer for the mixed-mode chromatography method of the present invention, both linear and step gradients of elution salt can be generated by mixing different percentages of two buffers with different concentrations of elution salt at different times. In one embodiment, the increasing concentration of elution salt in the mobile phase for the anion exchange chromatography method is a linear gradient from about 0 M to about 1 M. In another embodiment, the increasing concentration of elution salt in the mobile phase is a linear gradient from about 0.3 M to about 0.7 M. In yet another embodiment, the increasing concentration of elution salt in the mobile phase for the anion exchange chromatography method is a step gradient from about 0 M to about 1 M. In yet another embodiment, the increasing concentration of elution salt in the mobile phase is a step gradient from about 0.3 M to about 0.7 M.

[0074] In certain preferred embodiments, the mobile phase used for the anion exchange chromatography method of the present invention has a pH that increases over time, and the concentration of an elution salt in the mobile phase also increases over time. In other words, the mobile phase comprises a dual pH / salt gradient for separating a target oligonucleotide or carbohydrate-oligonucleotide conjugate compound from one or more impurities. As described in Example 3 herein, the use of a mobile phase comprising a dual pH / salt gradient resulted in improved separation of intact carbohydrate-oligonucleotide conjugate compounds (e.g., GalNAc-oligos) from unconjugated oligonucleotides and other impurities, compared to a mobile phase comprising only a salt gradient. Thus, in some embodiments, the mobile phase comprises a pH gradient of about 8.5 to about 11 and an elution salt gradient of about 0 M to about 1 M. In other embodiments, the mobile phase comprises a pH gradient of about 9.0 to about 10.5 and an elution salt gradient of about 0.3 M to about 0.7 M. In yet other embodiments, the mobile phase comprises a pH gradient of about 8.5 to about 10.5 and an elution salt gradient of about 0 M to about 0.8 M. A representative dual pH / salt gradient for the mobile phase for the anion exchange chromatography method of the present invention is set forth in the following table, where Buffer A has a pH of 8.5 and contains no elution salt (i.e., 0M), and Buffer B has a pH of 11 and contains 1M elution salt.

[0075] [Table 2]

[0076] Other possible gradients and methods for generating gradients for increasing the concentration of eluting salt and increasing pH of the mobile phase over a series of separations are known to those skilled in the art.

[0077] In some embodiments of the anion exchange chromatography method of the present invention, the mobile phase comprises about 10 mM to about 200 mM buffer, about 1% (v / v) to about 50% (v / v) organic solvent, and an eluting salt, the concentration of which is increased over time at a gradient of about 0 M to about 1 M, and the pH of the mobile phase is increased to about 8.5 to about 11. In other embodiments, the mobile phase comprises about 20 mM to about 100 mM buffer, about 1% (v / v) to about 20% (v / v) organic solvent, and an eluting salt, the concentration of which is increased over time at a gradient of about 0 M to about 1 M, and the pH of the mobile phase is increased to about 8.5 to about 11. For any of these mobile phase compositions, the buffer can be sodium phosphate, the organic solvent can be acetonitrile or methanol, and the eluting salt can be sodium chloride, potassium chloride, or ammonium chloride. For example, in certain embodiments, the mobile phase comprises about 10 mM to about 200 mM sodium phosphate buffer, about 1% (v / v) to about 50% (v / v) acetonitrile, and sodium chloride, and the concentration of sodium chloride is increased over time at a gradient of about 0 M to about 1 M, and the pH of the mobile phase is increased from about pH 8.5 to about 11. In some embodiments, the mobile phase comprises about 20 mM to about 100 mM sodium phosphate buffer, about 1% (v / v) to about 20% (v / v) acetonitrile, and sodium chloride, and the concentration of sodium chloride is increased over time at a gradient of about 0 M to about 1 M, and the pH of the mobile phase is increased from about pH 8.5 to about 11. In another embodiment, the mobile phase comprises about 15 mM to about 25 mM sodium phosphate buffer, about 12% (v / v) to about 18% (v / v) acetonitrile, and sodium chloride, wherein the sodium chloride concentration is increased over time at a gradient of about 0 M to about 1 M, and the pH of the mobile phase is increased from about 8.5 to about 11. In yet another embodiment, the mobile phase comprises about 15 mM to about 25 mM sodium phosphate buffer, about 12% (v / v) to about 18% (v / v) acetonitrile, and sodium chloride, wherein the sodium chloride concentration is increased over time at a gradient of about 0 M to about 0.8 M, and the pH of the mobile phase is increased from about 8.5 to about 10.5. In one embodiment, the mobile phase comprises about 20 mM sodium phosphate buffer, about 15% (v / v) acetonitrile, and sodium chloride, and the concentration of sodium chloride is increased over time in a gradient from about 0.3 M to about 0.7 M, and the pH of the mobile phase is increased from about pH 9.0 to about 10.5.In these and other embodiments, the acetonitrile concentration in the mobile phase can remain constant over time.

[0078] In certain embodiments of the anion exchange chromatography method of the present invention, the mobile phase comprises about 10 mM to about 200 mM sodium phosphate buffer, about 1% (v / v) to about 50% (v / v) methanol, and sodium chloride, wherein the concentration of sodium chloride is increased over time at a gradient of about 0 M to about 1 M, and the pH of the mobile phase is increased from about pH 8.5 to about 11. In some embodiments, the mobile phase comprises about 20 mM to about 100 mM sodium phosphate buffer, about 1% (v / v) to about 20% (v / v) methanol, and sodium chloride, wherein the concentration of sodium chloride is increased over time at a gradient of about 0 M to about 1 M, and the pH of the mobile phase is increased from about pH 8.5 to about 11. In another embodiment, the mobile phase comprises about 15 mM to about 25 mM sodium phosphate buffer, about 12% (v / v) to about 18% (v / v) methanol, and sodium chloride, with the sodium chloride concentration increasing over time at a gradient of about 0 M to about 1 M, and the mobile phase pH increasing from about 8.5 to about 11. In yet another embodiment, the mobile phase comprises about 15 mM to about 25 mM sodium phosphate buffer, about 12% (v / v) to about 18% (v / v) methanol, and sodium chloride, with the sodium chloride concentration increasing over time at a gradient of about 0 M to about 0.8 M, and the mobile phase pH increasing from about 8.5 to about 10.5. In any of the above mobile phase buffers, potassium chloride or ammonium chloride may be used as the eluting salt instead of sodium chloride.

[0079] As a solution containing oligonucleotide / carbohydrate-oligonucleotide conjugate compounds and one or more impurities moves through an anion exchange matrix with a mobile phase described herein (e.g., a dual pH / salt gradient mobile phase), elution fractions are collected. UV absorption, e.g., at 260 nm, can be used to monitor the oligonucleotide content in the fractions. As shown by the chromatograms in Figures 9A-9C, when anion exchange chromatography is operated according to the methods of the present invention, carbohydrate-oligonucleotide conjugate compounds (e.g., GalNAc-oligo) are eluted from the anion exchange matrix before unconjugated oligonucleotides (e.g., impurities in this content), thus allowing the collection of a set of fractions for carbohydrate-oligonucleotide conjugate compounds separated from one or more impurities. To verify the enrichment of fractions for carbohydrate-oligonucleotide conjugate compounds or other target oligonucleotides, samples from the elution fractions can be analyzed by gel electrophoresis, capillary electrophoresis, ion-pairing reversed-phase liquid chromatography-mass spectrometry, analytical ion exchange chromatography, and / or native mass spectrometry.

[0080] Separations using either a mixed-mode matrix or an anion-exchange matrix according to the methods of the invention can be performed at temperatures between about 5°C and about 45°C. In certain embodiments, separations using either a mixed-mode matrix or anion-exchange matrix according to the methods of the invention are performed at ambient temperature. For example, in some embodiments, separations using a mixed-mode matrix or anion-exchange matrix are performed at temperatures between about 15°C and about 25°C. In other embodiments, separations using a mixed-mode matrix or anion-exchange matrix are performed at temperatures between about 18°C ​​and about 22°C. In still other embodiments, separations using a mixed-mode matrix or anion-exchange matrix are performed at temperatures between about 20°C and about 25°C.

[0081] In certain embodiments of the mixed-mode chromatography or anion exchange chromatography methods of the present invention, an elution fraction or set of elution fractions containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound can be isolated and, optionally, pooled for further processing. For example, the elution fractions containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound can be subjected to one or more additional purification steps, such as affinity separation (e.g., nucleic acid hybridization using sequence-specific reagents), an additional ion exchange chromatography step (e.g., using a different stationary phase), a mixed-mode chromatography step, reversed-phase chromatography, or size exclusion chromatography (e.g., using a desalting column). In some embodiments of the mixed-mode chromatography methods of the present invention, the method comprises isolating an elution fraction or set of elution fractions containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound and subjecting the elution fractions to anion exchange chromatography, such as the anion exchange chromatography methods of the present invention described herein. In some embodiments of the anion exchange chromatography methods of the present invention, the method comprises isolating an elution fraction or set of elution fractions containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound and subjecting the elution fractions to mixed-mode chromatography, such as the mixed-mode chromatography methods of the present invention described herein.

[0082] As described in embodiments herein, the mixed-mode chromatography method of the present invention provides an orthogonal separation to the anion exchange chromatography method of the present invention. Thus, in certain embodiments, the two methods can be used in combination to achieve superior purification and obtain target oligonucleotides, particularly carbohydrate-oligonucleotide conjugate compounds. For example, in some embodiments, the anion exchange chromatography method is performed first, followed by the mixed-mode chromatography method. Thus, in certain embodiments, the method of the present invention comprises: contacting a solution containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities with an anion exchange matrix containing a strong anion exchange ligand; passing a first mobile phase through the anion exchange matrix, the first mobile phase having a pH of at least about 8.5 and comprising a buffer, an organic solvent, and an elution salt, wherein the concentration of the elution salt and the pH of the first mobile phase increase over time; collecting elution fractions from the anion exchange matrix containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound to generate an elution pool; contacting the elution pool with a mixed-mode matrix comprising a strong anion exchange ligand, a strong cation exchange ligand, and a hydrophobic ligand; passing a second mobile phase having a pH of about 7.0 to about 8.5, the second mobile phase comprising a buffer, an organic solvent, and an elution salt, the concentrations of the elution salt and the organic solvent increasing over time, through the mixed-mode matrix; Recovering elution fractions containing purified target oligonucleotides or carbohydrate-oligonucleotide conjugate compounds from the mixed-mode matrix.

[0083] In other embodiments, the mixed-mode chromatography method is performed first, followed by the anion exchange chromatography method. Thus, in certain embodiments, the method of the present invention comprises: contacting a solution containing a target oligonucleotide or carbohydrate-oligonucleotide conjugate compound and one or more impurities with a mixed-mode matrix containing strong anion-exchange ligands, strong cation-exchange ligands, and hydrophobic ligands; passing a first mobile phase having a pH of about 7.0 to about 8.5, the first mobile phase comprising a buffer, an organic solvent, and an elution salt, the concentrations of the elution salt and the organic solvent increasing over time, through a mixed-mode matrix; collecting elution fractions from the mixed-mode matrix containing the target oligonucleotide or carbohydrate-oligonucleotide conjugate compound to generate an elution pool; contacting the elution pool with an anion exchange matrix containing a strong anion exchange ligand; passing a second mobile phase through the anion exchange matrix, the second mobile phase having a pH of at least about 8.5 and comprising a buffer, an organic solvent, and an elution salt, wherein the concentration of the elution salt and the pH of the second mobile phase increase over time; Recovering the elution fractions containing the purified target oligonucleotide or carbohydrate-oligonucleotide conjugate compound from the anion exchange matrix.

[0084] Elution fractions collected from either the mixed-mode matrix or the anion-exchange matrix containing target oligonucleotides or carbohydrate-oligonucleotide conjugate compounds according to the methods of the present invention can be subjected to other reactions to modify the structure of the oligonucleotides or carbohydrate-oligonucleotide conjugate compounds. For example, in embodiments where the oligonucleotides or carbohydrate-oligonucleotide conjugate compounds are therapeutic molecules (e.g., antisense oligonucleotides) or components of therapeutic molecules (e.g., double-stranded RNA interference agents, e.g., siRNAs), the purified oligonucleotides or carbohydrate-oligonucleotide conjugate compounds in the elution fractions can be formulated into pharmaceutical compositions with pharmaceutically acceptable excipients for administration to patients for therapeutic purposes. In embodiments where target oligonucleotides are to be purified, the purified oligonucleotides in the elution fractions can be subjected to conjugation reactions to covalently attach targeting ligands, such as carbohydrate-containing ligands (e.g., to form carbohydrate-oligonucleotide conjugate compounds), cholesterol, or antibodies, to the oligonucleotides. In other embodiments, the purified oligonucleotides in the elution fractions can be encapsulated in exosomes, liposomes, or other types of lipid nanoparticles. In embodiments in which the oligonucleotide or carbohydrate-oligonucleotide conjugate compound is a component of a double-stranded RNA interfering agent (e.g., either the sense or antisense strand of an siRNA molecule), the purified oligonucleotide or carbohydrate-oligonucleotide conjugate compound in the elution fraction may be subjected to an annealing reaction to hybridize the oligonucleotide or carbohydrate-oligonucleotide conjugate compound to its complementary strand to form a double-stranded RNA interfering agent.

[0085] The methods of the present invention provide substantially pure preparations of target oligonucleotide or carbohydrate-oligonucleotide conjugate compounds. For example, in some embodiments, the purity of the oligonucleotide or carbohydrate-oligonucleotide conjugate compound in the elution fraction from the mixed-mode matrix or anion exchange matrix is ​​at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In certain embodiments, the purity of the oligonucleotide or carbohydrate-oligonucleotide conjugate compound in the elution fraction from the mixed-mode matrix or anion exchange matrix is ​​at least 90%. In other embodiments, the purity of the oligonucleotide or carbohydrate-oligonucleotide conjugate compound in the elution fraction from the mixed-mode matrix or anion exchange matrix is ​​at least 92%. In still other embodiments, the purity of the oligonucleotide or carbohydrate-oligonucleotide conjugate compound in the elution fraction from the mixed-mode matrix or anion exchange matrix is ​​at least 94%. Methods for detecting and quantifying oligonucleotides are known to those skilled in the art and can include ion-pairing reversed-phase liquid chromatography-mass spectrometry and analytical ion exchange methods, such as those described in the Examples.

[0086] The following examples, including the experiments conducted and results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. [Example]

[0087] Example 1. Purification of GalNAc-conjugated oligonucleotides using mixed-mode chromatography Ion exchange chromatography has become one of the key chromatographic techniques for the purification of oligonucleotides, given that oligonucleotide molecules contain many charge groups and are largely hydrophilic in nature. As a result, classic ion exchange supports such as Source Q15 or Q30 (GE Healthcare) and TSKgel SuperQ-5PW (Tosoh Bioscience) are often used for routine purification of oligonucleotides. These supports share very similar surface chemistries, incorporating quaternary amines and providing strong ion exchange interactions. However, as the field of nucleic acid therapeutics evolves and these therapeutic molecules become more complex due to chemical modifications to the oligonucleotide structure, ion exchange chromatography may not be adequate to achieve the desired recovery and purity of these modified oligonucleotides. This example describes the evaluation of a mixed-mode stationary phase containing both strong anion and cation exchange ligands and hydrophobic ligands for the purification of carbohydrate-conjugated oligonucleotides.

[0088] Various oligonucleotides containing 2'-O-methyl- and 2'-fluoro-modified nucleotides at either the 5'-terminal nucleotide via an aminohexyl linker or the 3'-terminal nucleotide via a homoserinyl linker were conjugated to triantennary N-acetylgalactosamine (GalNAc)-containing ligands (Structure 1). The structure of the triantennary GalNAc-containing ligand is shown below in Structure 1, where "Ac" represents an acetyl group and " / / " represents the point of attachment to the oligonucleotide via the aminohexyl or homoserinyl linker. [ka]

[0089] The oligonucleotides were synthesized on solid support using phosphoramidite chemistry. Table 1 below summarizes the sequences of the oligonucleotides. Nucleotide sequences are listed according to the following notation: dT, dA, dG, dC = corresponding deoxyribonucleotides; a, u, g, and c = corresponding 2'-O-methylribonucleotides; A, U, G, and C = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; Phos = terminal nucleotide with a monophosphate group at its 5' end; invAb = inverted abasic nucleotide (i.e., an abasic nucleotide linked to an adjacent nucleotide via a substitution at its 3' position (3'-3' linkage)); and invdX = inverted deoxyribonucleotide (i.e., a deoxyribonucleotide linked to an adjacent nucleotide via a substitution at its 3' position (3'-3' linkage)). The insertion of "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothiodiester group (e.g., a phosphorothioate internucleotide linkage). Unless otherwise indicated, all other nucleotides are linked by 3'-5' phosphodiester groups.

[0090] [Table 3]

[0091] [Table 4]

[0092] First, four different GalNAc-conjugated oligonucleotides (compound numbers 47-04, 40-07, 40-04, and 40-01) were purified using a conventional polymer bead-based strong anion exchange resin (TSKgel SuperQ-5PW, Tosoh Bioscience). Two to three milliliters of each GalNAc-oligonucleotide solution was loaded onto a TSKgel Super Q-5PW column (21.5 x 150 mm, 13 µm). Separation was performed using a salt gradient generated by mixing buffer A (20 mM NaHPO, 10% acetonitrile (v / v), pH 8.5) and buffer B (20 mM NaHPO, 10% acetonitrile (v / v), 1 M NaBr, pH 8.5). The gradient conditions were as follows: 0-20% buffer B in 0-3 min, 20-55% buffer B in 3-38 min, 55-65% buffer B in 38-44 min, and 20% buffer B in 45-55 min. The buffers were applied to the column at a flow rate of 8 mL / min, and the separation was performed at ambient temperature. The results of the separation are shown in Figure 1. The elution order for all four GalNAc-conjugated oligonucleotides is the intact GalNAc-conjugated oligonucleotide (Box 1), followed by the unconjugated oligonucleotide (Box 2). The later elution peak (Box 3) corresponds to a higher-order structure of the oligonucleotide resulting from secondary interactions. A similar profile has been reported for carbohydrate-conjugated oligonucleotides purified using a Resource Q ion-exchange column (GE Healthcare), suggesting that both columns provide the same elution order and similar selectivity. See Zhu and Mahato, Bioconjugate Chemistry, Vol. 21:2119-2127, 2010.

[0093] In an effort to improve the separation between GalNAc-conjugated and unconjugated oligonucleotides, a mixed-mode stationary phase was used. The Scherzo family of columns, with stationary phases containing functional groups that allow both ion exchange and hydrophobic interactions, is commercially available (Imtakt USA, Portland, OR). Three different Scherzo columns are available: SW-C18, SM-C18, and SS-C18. These columns differ in their ion exchange capacity, with either weak (ionizable) or strong (permanently charged) ion exchange functional groups, but all contain reversed-phase C18 (octadecyl) groups for hydrophobic interactions. The Scherzo SM-C18 column is the only column with a stationary phase containing weak ion exchange functional groups, while the other two columns have permanently charged stationary phases. The ion exchange capacity between the columns also differs, with the Scherzo SS-C18 column having the highest ion exchange capacity, followed by the SM-C18 column and then the SW-C18 column. See Biba et al., Journal of Chromatography A, Vol. 1304:69-77, 2013; imtaktusa.com / / wp-content / uploads / 2015 / 04 / Scherzo-Family-SS0.pdf. A Scherzo SS-C18 column was selected for the purification of GalNAc-conjugated oligonucleotides. The SS-C18 stationary phase contains strong ionic ligands (quaternary ammonium and sulfonyl groups) and C18 ligands. See, e.g., Choi et al., Forensic Science International, Vol. 259:69-76, 2016.

[0094] Two different GalNAc-conjugated oligonucleotides (compound numbers 40-01 and 09-01) were separated using a Scherzo SS-C18 analytical column (4.6 x 50 mm, 3 μm). A solution containing each GalNAc-conjugated oligonucleotide was loaded onto the column, and a salt / acetonitrile gradient was generated by mixing buffer A (100 mM Tris, pH 7.5) and buffer B (100 mM Tris, 20% acetonitrile (v / v), 1 M NaBr, pH 7.5). Separation was performed using the following gradient conditions: 40–70% buffer B from 0–20 min, with a hold of 40% buffer B from 20.1 min to 25.1 min. The buffer was applied to the column at a flow rate of 1.5 mL / min, and the separation was performed at ambient temperature. The results are shown in Figure 2. When compared with the TSKgel anion-exchange column, a reversal of the elution order was observed on the Scherzo SS-C18 mixed-mode column, with the more hydrophobic GalNAc-conjugated oligonucleotides being retained on the column longer than the unconjugated oligonucleotides. Thus, the Scherzo SS-C18 mixed-mode column allows for orthogonal separations relative to the TSKgel and Resource Q anion-exchange columns. Furthermore, the chromatogram peak for the separation of compound No. 40-01 (trace A in Figure 2) resembles a doublet, indicating possible separation of diastereomers. Note that compound No. 40-01 contains two phosphorothioate internucleotide linkages, thus creating a chiral center, while compound No. 09-01 does not contain phosphorothioate internucleotide linkages. Separation of diastereomers on the Scherzo SS-C18 mixed-mode column is described in more detail in Example 2.

[0095] Interestingly, the Scherzo mixed-mode column has previously been evaluated for analytical-scale separation of oligonucleotides. See Biba et al., Journal of Chromatography A, Vol. 1304:69-77, 2013. Although Biba et al. conclude that both Scherzo SW-C18 and SM-C18 columns are capable of separating oligoribonucleotides from their shortened versions using a mobile phase containing a salt gradient, the authors state that the Scherzo SS-C18 column was deemed unsuitable for oligonucleotide analysis, given that increasing the mobile phase strength did not result in elution of any oligonucleotides from the column. See page 77, left column, penultimate paragraph of Biba et al. The inventors discovered that the reversed-phase mode of the column can be affected by increasing the amount of organic modifiers (e.g., acetonitrile) in the mobile phase to manipulate the retention of oligonucleotides on the column. As shown by the results in Figure 2, elution of oligonucleotides can be achieved using a Scherzo SS-C18 column by increasing the concentration of acetonitrile in mobile phase buffer B to 20% or greater in combination with approximately 0.5-1 M salt (e.g., NaCl or NaBr), thereby indicating that both ion exchange and reversed-phase interactions control the retention of oligonucleotides on the column.

[0096] Separate experiments were performed to directly compare the purification of GalNAc-conjugated oligonucleotide (compound no. 34-01) by a Scherzo SS-C18 mixed-mode column or a TSKgel Super Q-5PW anion-exchange column. A solution containing 14.25 mg of GalNAc-conjugated oligonucleotide was loaded onto either a Scherzo SS-C18 mixed-mode column (10 × 250 mm, 3 μm) or a TSKgel Super Q-5PW anion-exchange column (21.5 mm × 300 mm (2 × 150 mm columns), 13 μm). The separation conditions for the Scherzo SS-C18 column were as follows: the mobile phase was applied at a flow rate of 5 mL / min; mobile phase buffer A: 100 mM Tris, pH 7.5; mobile phase buffer B: 100 mM Tris, 20% acetonitrile (v / v), 1 M NaBr, pH 7.5; gradient conditions: 55–80% buffer B from 0–40 min, 80% buffer B from 40–50 min, and 55% buffer B from 50.1–70 min. The separation conditions for the TSKgel Super Q-5PW column were as follows: mobile phase was applied at a flow rate of 8.5 mL / min; mobile phase buffer A: 20 mM NaHPO, 15% acetonitrile (v / v), pH 8.5; mobile phase buffer B: 20 ​​mM NaHPO, 15% acetonitrile (v / v), 1 M NaCl, pH 11; gradient conditions: 0-40% buffer B from 0-7 min, 40-65% buffer B from 7-67 min, and 80% buffer B from 67-87 min. Separations were performed at ambient temperature for both columns. The results of the separations are shown in Figure 3, and the recovery and purity of GalNAc-conjugated oligonucleotides from each separation on the two different stationary phases are summarized in Table 2. The fractions indicated by the dotted box in Figure 3 were collected and desalted by size exclusion chromatography (HiPrep 26 / 10 desalting column; 26 × 100 mm, 90 μm particle size; mobile phase: 80:20 water:ethanol; flow rate 10 mL / min).

[0097] [Table 5]

[0098] The Scherzo SS-C18 column allowed for a faster separation with sharper peaks compared to the TSKgel Super Q-5PW column (Figure 3; compare trace I with trace II). Importantly, purification on the Scherzo SS-C18 column resulted in greater recovery of GalNAc-conjugated oligonucleotides than that obtained on the TSKgel Super Q-5PW column, with 73% of the conjugated oligonucleotides recovered on the SS-C18 column compared to only 35% on the Super Q-5PW column (Table 2). Separation on the Scherzo SS-C18 column also improved the purity of GalNAc-conjugated oligonucleotides, achieving 95% purity compared to 91% obtained on the Super Q-5PW column (Table 2). These results demonstrate that a 10 mm internal diameter (ID) semi-preparative mixed-mode support is capable of purifying sufficient quantities of GalNAc-conjugated oligonucleotides and offers the potential for even larger gram-scale purifications. Purification using mixed-mode chromatography provides an improved method for purifying carbohydrate-conjugated oligonucleotides when compared to anion exchange chromatography.

[0099] Next, to evaluate the applicability of the above-described mixed-mode chromatography purification method, various GalNAc-conjugated oligonucleotides with different structural features were tested. Compounds 13-10, 13-13, 13-07, 32-10, 32-07, 32-04, and 32-01 were purified using a Scherzo SS-C18 column (10 × 250 mm, 3 μm). The sequences and chemical modifications for these compounds are provided in Table 1 above. A solution containing each of the GalNAc-conjugated oligonucleotides was loaded onto the column, and separation was performed using a salt / acetonitrile gradient generated by mixing buffer A (100 mM Tris, pH 7.5) and buffer B (100 mM Tris, 20% acetonitrile (v / v), 1 M NaBr, pH 7.5) under the following gradient conditions: 55–80% buffer B from 0–40 min, 80% buffer B from 40–50 min, and 55% buffer B from 50.1–70 min. Buffer was applied to the column at a flow rate of 5 mL / min, and separation was carried out at ambient temperature. Fractions indicated by the dotted box in Figure 4A were collected, combined, and desalted by size-exclusion chromatography (HiPrep 26 / 10 desalting column; 26 × 100 mm, 90 μm particle size; mobile phase: 80:20 water:ethanol; flow rate: 10 mL / min). The combined fractions were analyzed by analytical ion-pairing reversed-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS) method (Waters Xbridge BEH OST C18 column, 2.1 × 50 mm, 1.7 μm particle size; 0.6 mL / min flow rate; temperature: 60 °C) to determine the purity of the GalNAc-conjugated oligonucleotides in the fractions. Preparative chromatograms for each of the GalNAc-conjugated oligonucleotides are shown in Figure 4A, while the ion-pairing reversed-phase liquid chromatogram for the final desalted sample is shown in Figure 4B. Most of the GalNAc-conjugated oligonucleotides eluted from the preparative mixed-mode column by 25 minutes (Figure 4A). The final purity and recovery for each of the compounds are summarized in Table 3. Final purity ranged from 92% to 94%, while recoveries ranged from 29% to 57%. Compared to the recoveries reported in Table 2, recoveries for this experiment were calculated after desalting, which typically resulted in sample losses in the 10-20% range.No deterioration in performance of the Scherzo SS-C18 column was observed upon injection of highly basic samples prepared in concentrated ammonia, although the operating pH range of this column as provided by the manufacturer is reported to be 1.5 to 8. The maintenance of column performance appeared to be the result of the use of a well-buffered solution for the mobile phase (e.g., containing 100 mM TRIS and adjusted to pH 7.5).

[0100] [Table 6]

[0101] In another set of experiments, the purification method using a Scherzo SS-C18 mixed-mode column was applied to two crude samples containing different GalNAc-conjugated oligonucleotides with initial purities ranging from 20 to 25%. Our previous experiments with such low purity starting samples of GalNAc-conjugated oligonucleotides suggested that methods based on anion exchange chromatography (e.g., using a TSKgel Super Q-5PW column) generally cannot provide purities greater than 90% and adequate recoveries. A solution containing each GalNAc-conjugated oligonucleotide (compound no. 19-04 or compound no. 19-07) was loaded onto a Scherzo SS-C18 column (10 × 250 mm, 3 μm) and separated using a salt / acetonitrile gradient generated by mixing buffer A (100 mM Tris, pH 7.5) and buffer B (100 mM Tris, 20% acetonitrile (v / v), 1 M NaBr, pH 7.5) under the following gradient conditions: 45–70% buffer B from 0–40 min, 70–80% buffer B from 40–45 min, 80% buffer B from 45–50 min, and 45% buffer B from 51–66 min. The buffers were applied to the column at a flow rate of 5 mL / min, and separation was performed at ambient temperature. The fractions indicated by the dotted box in Figure 5 were collected, combined, and desalted by size-exclusion chromatography (HiPrep 26 / 10 desalting column; 26 x 100 mm, 90 μm particle size; mobile phase: 80:20 water:ethanol; flow rate: 10 mL / min). The combined fractions were analyzed by an analytical ion-pairing reverse-phase HPLC-MS method (Waters Xbridge BEH OST C18 column, 2.1 x 50 mm, 1.7 μm particle size; 0.6 mL / min flow rate; temperature: 60°C) to determine the purity of the GalNAc-conjugated oligonucleotides in the fractions. The preparative chromatogram is shown in Figure 5, and a summary of the purity and recovery of the final desalted sample is provided in Table 4. As shown in Table 4, the final sample exhibited a purity of greater than 90%.

[0102] [Table 7]

[0103] Taken together, the results of the experiments described in this example demonstrate that the use of mixed-mode chromatography resulted in more than two-fold recovery and allowed for higher purity for GalNAc-conjugated oligonucleotides when compared to anion-exchange chromatography. The mixed-mode chromatography method, which proved suitable for purifying a variety of GalNAc-conjugated oligonucleotides when applied to conjugates with distinct structural features, resulted in high recoveries and purities in the 92-94% range.

[0104] Example 2. Separation of phosphorothioate diastereomers The mixed-mode stationary phase appeared to provide separation of the diastereomers, as observed in the previous experiment described in Example 1. To further explore this result, a solution containing Compound No. 08-17, which contains four phosphorothioate internucleotide linkages, thereby giving rise to four chiral centers, was separated using either a Scherzo SS-C18 mixed-mode analytical column (4.6 × 50 mm, 3 μm) or a TSKgel Super Q-5PW anion-exchange analytical column (7.5 mm × 75 mm, 10 μm). The separation conditions for the Scherzo SS-C18 analytical column were as follows: the mobile phase was applied at a flow rate of 1 mL / min; mobile phase buffer A: 100 mM Tris, pH 7.5; mobile phase buffer B: 100 mM Tris, 20% acetonitrile (v / v), 1 M NaBr, pH 7.5; gradient conditions: 55–80% buffer B from 0–8 min, 80% buffer B from 8–10 min, and 55% buffer B from 10.1–12 min. The separation conditions for the TSKgel Super Q-5PW analytical column were as follows: the mobile phase was applied at a flow rate of 2 mL / min; mobile phase buffer A: 20 mM NaHPO, 15% acetonitrile (v / v), pH 8.5; mobile phase buffer B: 20 ​​mM NaHPO, 15% acetonitrile (v / v), 1 M NaCl, pH 11; gradient conditions: 0-45% buffer B from 0-0.75 min, 45-80% buffer B from 0.75-6.00 min, 80-100% buffer B from 6.00-6.10 min, 100% buffer B from 6.10-7.00 min, and 0% buffer B at 7.10 min. For both columns, separations were performed at ambient temperature. The separation results are shown in Figure 6, which reveals that the mixed-mode stationary phase provides superior separation when compared to the anion-exchange stationary phase, as evidenced by the presence of multiple peaks. The multiple peaks may correspond to the separation of diastereomers.

[0105] Next, a sample containing the same compounds 08-17 was purified using a semi-preparative Scherzo SS-C18 mixed-mode column. The sample was loaded onto a Scherzo SS-C18 column (10 × 250 mm, 3 μm) and separated using a salt / acetonitrile gradient generated by mixing buffer A (100 mM Tris, pH 7.5) and buffer B (100 mM Tris, 20% acetonitrile (v / v), 1 M NaBr, pH 7.5) using the following gradient conditions: 45–70% buffer B from 0–40 min, 70–80% buffer B from 40–45 min, 80% buffer B from 45–50 min, and 45% buffer B from 51–66 min. The buffers were applied to the column at a flow rate of 5 mL / min, and the separation was performed at ambient temperature. The resulting preparative chromatogram is shown in Figure 7A. The peak profile is similar to that obtained on the analytical column (Figure 6), with substantially improved separation, primarily due to the extended length of the semi-preparative column. The peaks labeled 1, 2, and 3 in Figure 7A were collected as individual fractions, desalted by size-exclusion chromatography (HiPrep 26 / 10 desalting column; 26 × 100 mm, 90 μm particle size; mobile phase: 80:20 water:ethanol; flow rate: 10 mL / min), and analyzed by analytical ion-pairing reversed-phase HPLC-MS (Waters Xbridge BEH OST C18 column, 2.1 × 50 mm, 1.7 μm particle size; 0.6 mL / min flow rate; temperature: 60 °C) to confirm the identity of the test substance in each fraction. The ion-pairing reversed-phase liquid chromatograms for each of the three fractions are shown in Figure 7B. Each fraction contains primarily a single peak, exhibiting greater than 90% purity and comparable m / z values ​​(Figure 7B and data not shown). The single peak still likely corresponds to a mixture of diastereomers. Nevertheless, the Scherzo SS-C18 mixed-mode column offers a novel approach for better separation of phosphorothioate diastereomers on a preparative scale.

[0106] In separate experiments, to determine whether diastereomeric separation could be observed again, a Scherzo SS-C18 mixed-mode semi-preparative column was used to purify six different GalNAc-conjugated oligonucleotides, each with two phosphorothioate internucleotide linkages but with different structures. Compounds 24-10, 24-13, 24-16, 24-19, 24-22, and 24-25 were purified using a Scherzo SS-C18 column (10 × 250 mm, 3 μm). The sequences and chemical modifications for these compounds are provided in Table 1 above. A solution containing each GalNAc-conjugated oligonucleotide was loaded onto the column and separated using a salt / acetonitrile gradient generated by mixing buffer A (100 mM Tris, pH 7.5) and buffer B (100 mM Tris, 20% acetonitrile (v / v), 1 M NaBr, pH 7.5) under the following gradient conditions: 55–80% buffer B from 0–40 min, 80% buffer B from 40–50 min, and 55% buffer B from 50.1–70 min. The buffer was applied to the column at a flow rate of 5 mL / min, and separation was performed at ambient temperature. The fractions indicated by the dotted box in Figure 8A were collected, combined, and desalted by size-exclusion chromatography (HiPrep 26 / 10 desalting column; 26 × 100 mm, 90 μm particle size; mobile phase: 80:20 water:ethanol; flow rate: 10 mL / min). To determine the purity of the GalNAc-conjugated oligonucleotides in the fractions, the combined fractions were analyzed by analytical ion-pairing reverse-phase HPLC-MS (Waters Xbridge BEH OST C18 column, 2.1 × 50 mm, 1.7 μm particle size; 0.6 mL / min flow rate; temperature 60°C). The preparative chromatogram for each of the GalNAc-conjugated oligonucleotides is shown in Figure 8A, while the ion-pairing reverse-phase liquid chromatogram for the final desalted sample is shown in Figure 8B. The final purity and recovery for each of the compounds are summarized in Table 5.

[0107] [Table 8]

[0108] The final purity of this compound was greater than 90%, with recoveries ranging from 28% to 40%, consistent with the purity and recoveries for structurally distinct GalNAc-conjugated oligonucleotides using this stationary phase (see Table 3). Separation of the phosphorothioate diastereomers was observed, as indicated by multiple peaks in the preparative chromatogram shown in Figure 8A. Diastereomeric separation is further evident in the analytical chromatogram of isolated fractions from the preparative purification shown in Figure 8B. A doublet of peaks corresponding to analytes with the same m / z value was observed. Although only the fractions shown in the dotted box in Figure 8A were combined, nearby peaks adjacent to the combined fractions also revealed the same m / z value, indicating the presence of diastereomers. Although the semi-preparative Scherzo SS-C18 column does not provide baseline separation of all possible phosphorothioate diastereomers, it does provide a significant improvement in diastereomeric separation when compared to methods based on anion exchange chromatography, such as those using a TSKgel Super Q-5PW column.

[0109] Example 3. Purification of GalNAc-conjugated oligonucleotides using anion exchange chromatography This example describes an alternative method for separating carbohydrate-conjugated oligonucleotides using anion exchange chromatography. The use of a pH gradient to elute oligonucleotides from a weak anion exchange column has been previously reported (see Zimmermann et al., J. Chromatogr A, Vol. 1354:43-55, 2014). Elution occurred over a very narrow pH range of 7-8, and changes in elution and selectivity were largely attributed to changes in stationary phase ionization (Zimmermann et al., 2014). In contrast, the method described in this example utilizes a permanently charged stationary phase and a pH gradient of 8.5-11 to tailor the ionization of carbohydrate-conjugated oligonucleotides. It has been reported that increasing the pH promotes the ionization of G, T, and U bases, thereby increasing the overall negative charge of the oligonucleotide and affecting separation selectivity (McGinnis et al., J Chromatogr B, Vol. 883-884:76-94, 2012 and Thayer et al., J Chromatogr B, Vol. 878:933-941, 2010). It has also been reported that increasing the pH reduces secondary interactions, making ion-exchange purification more straightforward (McCarthy et al., J. Anal. Biochem., Vol. 390:181-188, 2009). To facilitate the separation of GalNAc-conjugated oligonucleotides from impurities, a dual pH / salt gradient elution was developed for a strong anion-exchange resin based on polymer beads (TSKgel SuperQ-5PW, Tosoh Bioscience).

[0110] A GalNAc-conjugated oligonucleotide (compound no. 34-01; see Table 1 for structural characteristics) was separated from impurities using a TSKgel SuperQ-5PW anion-exchange analytical column (7.5 × 75 mm, 10 μm) eluted with either a salt gradient at constant pH or a dual salt and pH gradient. Under the first set of conditions (Figure 9A, trace B), a solution containing GalNAc-conjugated oligonucleotides was loaded onto the column and separated using a salt gradient generated by mixing buffer A (20 mM NaHPO, 10% acetonitrile (v / v), pH 8.5) and buffer B (20 mM NaHPO, 10% acetonitrile (v / v), 1 M NaBr, pH 8.5) using the following gradient conditions: 30–60% Buffer B from 0.75–5.00 min; 60–65% Buffer B from 5.00–5.50 min; 65–70% Buffer B from 5.50–6.00 min; 70% Buffer B from 6.00–9.00 min; and 0% Buffer B at 9.1 min. Under the second set of conditions (Figure 9A, trace A), a solution containing GalNAc-conjugated oligonucleotides was loaded onto the column and separated using a dual pH / salt gradient generated by mixing buffer A (20 mM NaHPO, 10% acetonitrile (v / v), pH 8.5) and buffer B (20 mM NaHPO, 10% acetonitrile (v / v), 1 M NaBr, pH 11) using the same gradient parameters as described immediately above for the salt gradient. The buffers were applied to the column at a flow rate of 2 mL / min, and the separation was carried out at 40°C.

[0111] Next, the mobile phase components for the dual pH / salt gradient elution method were adjusted to optimize the separation of intact GalNAc-conjugated oligonucleotides from their unconjugated counterparts. Different salt and organic modifier (e.g., acetonitrile) concentrations in the mobile phase were evaluated. Specifically, the mobile phase buffers in each of the three sets of conditions were as follows: ·Condition 1: Buffer A1: 20 mM Na2HPO4, 10% acetonitrile (v / v), pH 8.5 〇 Buffer B1: 20mM Na2HPO4, 10% acetonitrile (v / v), 1M NaBr, pH11 ·Condition 2: 〇 Buffer A2: 20mM Na2HPO4, 10% acetonitrile (v / v), pH8.5 〇 Buffer B2: 20mM Na2HPO4, 10% acetonitrile (v / v), 1M NaCl, pH11 ·Condition 3: Buffer A3: 20 mM NaHPO, 15% acetonitrile (v / v), pH 8.5 Buffer B3: 20 mM Na2HPO4, 15% acetonitrile (v / v), 1 M NaCl, pH 11

[0112] A solution containing GalNAc-conjugated oligonucleotide (compound no. 34-01) was loaded onto a TSKgel SuperQ-5PW anion-exchange analytical column (7.5 × 75 mm, 10 μm) and separated at 25°C with a mobile phase flow rate of 2 mL / min using a dual pH / salt gradient generated by mixing the two buffers shown above for each of the following conditions:

[0113] [Table 9]

[0114] Figure 9B shows chromatograms for separations using different mobile phase buffers with different counter anions or organic modifier concentrations. A chloride counter anion in the mobile phase, combined with 15% acetonitrile, yielded the greatest selectivity, resulting in the best separation (Figure 9B, trace C) and purity in the range of 96-97% for gram-scale purification of this GalNAc-conjugated oligonucleotide (data not shown). Typical conditions for preparative purification are as follows: Column: TSKgel SuperQ-5PW (21.5 x 300 mm, 13 μm) ·Flow rate: 8.5mL / min Temperature: Ambient (e.g. 18°C ​​to 24°C) ·Mobile phase: Buffer A: 20 mM NaHPO, 15% (v / v) acetonitrile, pH 8.5 Buffer B: 20 ​​mM NaHPO, 15% (v / v) acetonitrile, 1 M NaCl, pH 11 · Gradient purification conditions.

[0115] [Table 10]

[0116] A solution containing Compound No. 34-01 was purified on a preparative scale using the same preparative conditions as described above. Specifically, two TSKgel SuperQ-5PW columns (each column: 21.5 × 150 mm, 13 μm) were connected in series, and the solution (1.2 mL) was loaded onto the first of the two connected columns. Separation was performed at ambient temperature with a mobile phase flow rate of 8.5 mL / min using a dual pH / salt gradient. A gradient was generated by mixing buffer A (20 mM NaHPO, 15% (v / v) acetonitrile, pH 8.5) and buffer B (20 mM NaHPO, 15% (v / v) acetonitrile, 1 M NaCl, pH 11) according to the following gradient conditions: 0-30% buffer B from 0-7 min, 30-65% buffer B from 7-63 min, 65-70% buffer B from 63-63.1 min, 70% buffer B from 63.1-66 min, and re-equilibration with buffer A (100%) from 66-80 min. The resulting preparative chromatogram is shown in Figure 9C. The peak profile is similar to that obtained using an analytical column (Figure 9B, trace C).

[0117] A summary of the parameters for the analytical ion-pairing reversed-phase chromatography and desalting methods described in the Examples is provided below. Analytical ion-pairing reversed-phase liquid chromatography-mass spectrometry Column: Waters Xbridge BEH OST C18 (2.1 x 50 mm, 1.7 μm) ·Flow rate: 0.6mL / min ·Temperature: 60℃ ·Mobile phase: Buffer A: 15.7 mM N,N-diisopropylethylamine (DIEA), 50 mM hexafluoro-2-propanol (HFIP) in water Buffer B: 15.7 mM DIEA, 50 mM HFIP:acetonitrile (50:50) in water · Gradient purification conditions.

[0118] [Table 11]

[0119] Detection: Diode array detector at 260 nm followed by mass spectrometry Desalting (size exclusion chromatography) Column: HiPrep 26 / 10 desalting column (26 x 100 mm, 90 μm) Mobile phase: 80:20 water:ethanol ·Flow rate=10mL / min

[0120] All publications, patents, and patent applications discussed and cited herein are hereby incorporated by reference in their entirety. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0121] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for purifying a carbohydrate-oligonucleotide conjugate compound from one or more impurities, comprising: contacting a solution containing the carbohydrate-oligonucleotide conjugate compound and one or more impurities with a mixed-mode matrix comprising a strong anion exchange ligand, a strong cation exchange ligand, and a hydrophobic ligand; passing a mobile phase having a pH of about 7.0 to about 8.5, the mobile phase comprising a buffer, an organic solvent, and an elution salt, the concentration of the elution salt and the organic solvent increasing over time, through the mixed-mode matrix; collecting elution fractions from the mixed-mode matrix, wherein one or more impurities are eluted in a first set of elution fractions and the carbohydrate-oligonucleotide conjugate compound is eluted in a second set of elution fractions, thereby separating the carbohydrate-oligonucleotide conjugate compound from the impurities. method.

2. The method of claim 1 , wherein the strong anion exchange ligand comprises a quaternary amine.

3. The method of claim 1 , wherein the strong cation exchange ligand comprises a sulfonyl functional group.

4. The method of claim 1 , wherein the hydrophobic ligand comprises an alkyl group.

5. The method of claim 4 wherein the alkyl group comprises an octadecyl carbon chain.

6. The method of claim 1 , wherein the mixed-mode matrix has a pore size of less than 20 nm.

7. The method of claim 1 , wherein the mixed-mode matrix has a pore size of about 8 nm to about 15 nm.

8. 2. The method of claim 1, wherein the buffer is sodium phosphate, Tris hydrochloride, HEPES, or MOPS.

9. 2. The method of claim 1, wherein the organic solvent is acetonitrile, methanol, propanol, isopropanol, ethanol, butanol, tetrahydrofuran, or acetone.

10. 2. The method of claim 1, wherein the increasing concentration of the organic solvent in the mobile phase is a gradient from about 8% (v / v) to about 20% (v / v).

11. 2. The method of claim 1, wherein the increasing concentration of the organic solvent in the mobile phase is a gradient from about 10% (v / v) to about 18% (v / v).

12. 12. The method of claim 10 or claim 11, wherein the gradient is a step gradient.

13. 12. The method of claim 10 or claim 11, wherein the gradient is a linear gradient.

14. 2. The method of claim 1, wherein the cation in the eluting salt is sodium, potassium, ammonium, trimethylammonium, or triethylammonium.

15. 2. The method of claim 1, wherein the anion in the eluting salt is chloride, bromide, nitrate, nitrite, iodide, perchlorate, acetate, or formate.

16. 2. The method of claim 1, wherein the eluting salt is sodium bromide, potassium bromide, ammonium bromide, sodium chloride, potassium chloride, or ammonium chloride.

17. 17. The method of claim 16, wherein the eluting salt is sodium bromide.

18. 2. The method of claim 1, wherein the increasing concentration of the eluting salt in the mobile phase is a gradient from about 0.5 M to about 1 M.

19. 20. The method of claim 18, wherein the gradient is a step gradient.

20. 19. The method of claim 18, wherein the gradient is a linear gradient.

21. 2. The method of claim 1, wherein the mobile phase has a pH of about 7.0 to about 8.0 and comprises about 20 mM to about 200 mM Tris-hydrochloride buffer, acetonitrile, and sodium bromide, wherein the concentration of acetonitrile is increased over time with a gradient of about 8% (v / v) to about 20% (v / v), and the concentration of sodium bromide is increased over time with a gradient of about 0.5 M to about 1 M.

22. 22. The method of claim 21, wherein the mobile phase has a pH of about 7.5 and comprises about 100 mM Tris-hydrochloride buffer, acetonitrile, and sodium bromide, wherein the concentration of acetonitrile is increased over time in a gradient from about 11% (v / v) to about 17% (v / v), and the concentration of sodium bromide is increased over time in a gradient from about 0.5 M to about 0.85 M.

23. 23. The method of any one of claims 1 to 22, wherein the carbohydrate in the carbohydrate-oligonucleotide conjugate compound comprises one or more hexose or hexosamine units.

24. 24. The method of any one of claims 1 to 23, wherein the carbohydrate in the carbohydrate-oligonucleotide conjugate compound comprises one or more galactose, galactosamine, or N-acetyl-galactosamine units.

25. 25. The method of any one of claims 1 to 24, wherein the carbohydrate in the carbohydrate-oligonucleotide conjugate compound comprises a multivalent galactose moiety or a multivalent N-acetyl-galactosamine moiety.

26. 26. The method of claim 25, wherein the multivalent galactose or N-acetyl-galactosamine moiety is trivalent or tetravalent.

27. The method of any one of claims 1 to 26, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound comprises at least one modified nucleotide.

28. 28. The method of claim 27, wherein the modified nucleotide is a 2'-modified nucleotide.

29. 28. The method of claim 27, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), or a combination thereof.

30. 30. The method of any one of claims 1 to 29, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound comprises at least one phosphorothioate internucleotide linkage.

31. 31. The method of claim 30, wherein the solution containing the carbohydrate-oligonucleotide conjugate compound further comprises one or more phosphorothioate diastereomers of the conjugate compound, and wherein a first diastereomer is eluted in a different, distinct set of elution fractions from a second diastereomer.

32. 32. The method of any one of claims 1 to 31, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound is from about 10 nucleotides to about 50 nucleotides in length.

33. 33. The method of claim 32, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound is from about 15 nucleotides to about 30 nucleotides in length.

34. 34. The method of any one of claims 1 to 33, further comprising isolating a set of elution fractions containing said carbohydrate-oligonucleotide conjugate compound.

35. 35. The method of claim 34, further comprising subjecting the fraction containing the carbohydrate-oligonucleotide conjugate compound to anion exchange chromatography.

36. 35. The method of any one of claims 1 to 34, wherein the solution containing the carbohydrate-oligonucleotide conjugate compound and one or more impurities is an eluate from an anion exchange chromatography matrix.

37. 1. A method for purifying a carbohydrate-oligonucleotide conjugate compound from one or more impurities, comprising: contacting a solution containing the carbohydrate-oligonucleotide conjugate compound and one or more impurities with an anion exchange matrix containing a strong anion exchange ligand; passing a mobile phase having a pH of at least about 8.5, the mobile phase comprising a buffer, an organic solvent, and an elution salt, the concentration of the elution salt and the pH of the mobile phase increasing over time, through the anion exchange matrix; collecting elution fractions from the anion exchange matrix, wherein the carbohydrate-oligonucleotide conjugate compound is eluted in a first set of elution fractions and one or more impurities are eluted in a second set of elution fractions, thereby separating the carbohydrate-oligonucleotide conjugate compound from the impurities.

38. 38. The method of claim 37, wherein the strong anion exchange ligand comprises a quaternary amine.

39. 38. The method of claim 37, wherein the buffer is sodium phosphate.

40. 38. The method of claim 37, wherein the organic solvent is acetonitrile, methanol, propanol, isopropanol, ethanol, butanol, tetrahydrofuran, or acetone.

41. 38. The method of claim 37, wherein the concentration of the organic solvent in the mobile phase is from about 1% (v / v) to about 20% (v / v).

42. 38. The method of claim 37, wherein the cation in the eluting salt is sodium, potassium, ammonium, trimethylammonium, or triethylammonium.

43. 38. The method of claim 37, wherein the anion in the eluting salt is chloride, bromide, nitrate, nitrite, iodide, perchlorate, acetate, or formate.

44. 38. The method of claim 37, wherein the eluting salt is sodium bromide, potassium bromide, ammonium bromide, sodium chloride, potassium chloride, or ammonium chloride.

45. 45. The method of claim 44, wherein the eluting salt is sodium chloride.

46. 38. The method of claim 37, wherein the increasing concentration of the eluting salt in the mobile phase is a gradient from about 0 M to about 1 M.

47. 38. The method of claim 37, wherein the increasing concentration of the eluting salt in the mobile phase is a gradient from about 0.3 M to about 0.7 M.

48. 38. The method of claim 37, wherein the pH of the mobile phase is increased from about 8.5 to about 11.

49. 38. The method of claim 37, wherein the pH of the mobile phase is increased to a pH of from about 9.0 to about 10.

5.

50. 38. The method of claim 37, wherein the mobile phase comprises about 20 mM to about 100 mM sodium phosphate buffer, about 1% (v / v) to about 20% (v / v) acetonitrile and sodium chloride, and the concentration gradient of sodium chloride is increased over time with a gradient from about 0 M to about 1 M, and the pH of the mobile phase is increased from about pH 8.5 to about 11.

51. 51. The method of claim 50, wherein the mobile phase comprises about 20 mM sodium phosphate buffer, about 15% (v / v) acetonitrile and sodium chloride, and the concentration of sodium chloride is increased over time in a gradient from about 0.3 M to about 0.7 M, and the pH of the mobile phase is increased from about pH 9.0 to about 10.

5.

52. 52. The method of any one of claims 37 to 51, wherein the carbohydrate in the carbohydrate-oligonucleotide conjugate compound comprises one or more hexose or hexosamine units.

53. 53. The method of any one of claims 37 to 52, wherein the carbohydrate in the carbohydrate-oligonucleotide conjugate compound comprises one or more galactose, galactosamine, or N-acetyl-galactosamine units.

54. 54. The method of any one of claims 37 to 53, wherein the carbohydrate in the carbohydrate-oligonucleotide conjugate compound comprises a multivalent galactose moiety or a multivalent N-acetyl-galactosamine moiety.

55. 55. The method of claim 54, wherein the multivalent galactose moiety or multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent.

56. 56. The method of any one of claims 37 to 55, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound comprises at least one modified nucleotide.

57. 57. The method of claim 56, wherein the modified nucleotide is a 2'-modified nucleotide.

58. 57. The method of claim 56, wherein the modified nucleotides are 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof.

59. 59. The method of any one of claims 37 to 58, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound comprises at least one phosphorothioate internucleotide linkage.

60. 60. The method of any one of claims 37 to 59, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound is from about 10 nucleotides to about 50 nucleotides in length.

61. 61. The method of claim 60, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound is from about 15 nucleotides to about 30 nucleotides in length.

62. 62. The method of any one of claims 37 to 61, further comprising isolating a set of elution fractions containing said carbohydrate-oligonucleotide conjugate compound.

63. 63. The method of claim 62, further comprising subjecting the fraction containing the carbohydrate-oligonucleotide conjugate compound to mixed-mode chromatography.

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