Improved Purification of GalNAc-Conjugated Antisense Oligonucleotides Using Boronic Acids

Enhanced purification of GalNAc-conjugated oligonucleotides has been realized using boronic acids as mobile phase additives during reversed-phase chromatography. Several substituted phenylboronic acids (PBAs) and alkyl boronic acids have been evaluated for resolution of GalNAc-conjugated and -unconjugated antisense oligonucleotides across a mobile phase pH range of 7-12. The boronic acid additives work by reversibly binding to sugar alcohol groups in the conjugate molecules, thus increasing their hydrophobicity and affinity for the stationary phase. Several combinations of boronic acid, additive concentration, and pH were evaluated with some demonstrating substantial improvements in resolution over additive-free control purifications. The best performing boronic acids in a model system were PBA at pH 8, 2-methylphenylboronic acid (2-MePBA) at pH 9, and 2-trifluoromethylphenylboronic acid (2-CF(3)PBA) at pH >= 10. In addition to separating GalNAc-conjugated from unconjugated oligonucleotides efficiently, complete resolution of a common GalNAc-containing branchmer impurity could be achieved. The boronic acid additives could be efficiently removed from the product fraction by ethanol precipitation, membrane ultrafiltration, or rinsing from the purification column prior to product elution.

Automated summary

This study demonstrates the enhanced purification of GalNAc-conjugated oligonucleotides using boronic acids as mobile phase additives during reversed-phase chromatography. The boronic acid additives work by increasing the hydrophobicity and affinity of the conjugate molecules for the stationary phase. The best performing boronic acids were PBA at pH 8, 2-MePBA at pH 9, and 2-CF(3)PBA at pH >= 10.