Purification methods for carbohydrate-linked oligonucleotides

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.