# US 12,760,057 B2 · Purification methods for carbohydrate-linked oligonucleotides

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- Number: US 12,760,057 B2
- Document ID: US12760057B2
- Status: Granted
- Kind: B2
- Application number: 17/620,645
- Filed: 2020-06-24
- Published: 2022-11-17
- Granted: 2026-10-13
- Earliest claimed: 2019-06-25
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Artaches Kazarian; Wesley Barnhart
- Web page: https://patentgrep.com/patent/US12760057B2

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- B01J 39/02: Processes using inorganic exchangers
- B01J 41/04: Processes using organic exchangers
- C07H 1/06: Separation; Purification
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

The present invention relates to methods for purifying nucleic acids. In particular, 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.

## Claims

### Claim 1

A method for purifying a carbohydrate-oligonucleotide conjugate compound from one or more impurities, comprising:
contacting a solution comprising the carbohydrate-oligonucleotide conjugate compound and one or more impurities with a mixed-mode matrix, wherein the mixed-mode matrix comprises a strong anion exchange ligand, a strong cation exchange ligand, and a hydrophobic ligand, wherein 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 octadecyl carbon chain;
passing a mobile phase through the mixed-mode matrix, wherein the mobile phase has a pH of about 7.0 to about 8.5 and comprises a buffer, acetonitrile, and an elution salt selected from the group consisting of sodium bromide, potassium bromide, ammonium bromide, sodium chloride, potassium chloride, and ammonium chloride, and wherein the concentration of acetonitrile in the mobile phase increases as a gradient from about 8% (v/v) to about 20% (v/v) and the concentration of the elution salt increases as a gradient from about 0.5 M to about 1 M; 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 carbohydrate-oligonucleotide conjugate compound is eluted in a second set of elution fractions, thereby separating the carbohydrate-oligonucleotide conjugate compound from the impurities, wherein the carbohydrate-oligonucleotide conjugate compound comprises a trivalent or tetravalent N-acetyl-galactosamine moiety covalently linked, optionally through a linker moiety, to an oligonucleotide that is about 10 nucleotides in length to about 50 nucleotides in length.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

The method of claim 1, wherein the increase in concentration of acetonitrile in the mobile phase is a gradient from about 10% (v/v) to about 18% (v/v).

### Claim 6 (depends on 1)

The method of claim 1, wherein the gradient of acetonitrile is a step gradient.

### Claim 7 (depends on 1)

The method of claim 1, wherein the gradient of acetonitrile is a linear gradient.

### Claim 8 (depends on 1)

The method of claim 1, wherein the elution salt is sodium bromide.

### Claim 9 (depends on 1)

The method of claim 1, wherein the gradient of the elution salt is a step gradient.

### Claim 10 (depends on 1)

The method of claim 1, wherein the gradient of the elution salt is a linear gradient.

### Claim 11 (depends on 1)

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, and wherein the concentration of acetonitrile increases at a gradient of about 8% (v/v) to about 20% (v/v) and the concentration of sodium bromide increases at a gradient of about 0.5 M to about 1 M over time.

### Claim 12 (depends on 11)

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

### Claim 13 (depends on 1)

The method of claim 1, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound comprises at least one modified nucleotide.

### Claim 14 (depends on 13)

The method of claim 13, wherein the modified nucleotide is a 2′-modified nucleotide.

### Claim 15 (depends on 13)

The method of claim 13, 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 combinations thereof.

### Claim 16 (depends on 1)

The method of claim 1, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound comprises at least one phosphorothioate internucleotide linkage.

### Claim 17 (depends on 16)

The method of claim 16, wherein the solution comprising the carbohydrate-oligonucleotide conjugate compound further comprises one or more phosphorothioate diastereomers of the conjugate compound, and wherein a first diastereomer elutes in a separate set of elution fractions than a second diastereomer.

### Claim 18 (depends on 1)

The method of claim 1, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound is about 15 nucleotides in length to about 30 nucleotides in length.

### Claim 19 (depends on 1)

The method of claim 1, further comprising isolating the set of elution fractions comprising the carbohydrate-oligonucleotide conjugate compound and subjecting the fractions comprising the carbohydrate-oligonucleotide conjugate compound to anion-exchange chromatography.

### Claim 20 (depends on 1)

The method of claim 1, wherein the solution comprising the carbohydrate-oligonucleotide conjugate compound and one or more impurities is an eluate from an anion-exchange chromatography matrix.

### Claim 21 (depends on 1)

The method of claim 1, wherein the N-acetyl-galactosamine moiety in the carbohydrate-oligonucleotide conjugate compound has the following structure:
wherein Ac represents an acetyl group and//represents the point of attachment, optionally through a linker, to the oligonucleotide.

### Claim 22 (depends on 1)

The method of claim 1, wherein the oligonucleotide in the carbohydrate-oligonucleotide conjugate compound comprises one or more 2′-fluoro modified nucleotides, 2′-O-methyl modified nucleotides, or combinations thereof.

### Claim 23 (depends on 1)

The method of claim 1, wherein the mixed-mode matrix comprises a density of the strong anion exchange ligand and the strong cation exchange ligand of 450 μmol/gram or greater.