Purification of antisense oligonucleotides using hydrophobic interaction chromatography

Abstract Hydrophobic interaction chromatography (HIC) provides a powerful alternative impurity control method for antisense oligonucleotide purification relative to traditionally used anion exchange (AEX) and/or reverse phase methods. HIC is particularly effective in clearing process‐related solvents and small molecules by ≥3 log 10 as well as failure sequences (sometimes called early eluting impurities (EEIs) by ≥90%). Additionally, HIC reduces harder to remove product‐related impurities. These include branchmers (late eluting impurities (LEIs), oligonucleotides missing a single nucleotide (N‐1 impurities), oligonucleotides lacking appropriate phosphorothioate sulfurization (P = O 1 impurity), and other synthesis‐related impurities. To optimize the purification process, variables such as resin ligand, salt types, processing conditions, types of gradients, and loading ratios were systematically evaluated to achieve 90% yield and maximal impurity resolution. Loading the column at 32%–78% of its dynamic binding capacity (DBC), combined with stepwise wash and elution gradients, provided effective resolution of impurities in crude oligonucleotide mixtures. The desorption of the purified product was achieved in low lyotropic salt concentrations (typically ≤50 mM) using a stepwise gradient. This approach retained non‐polar impurities such as LEIs within the column. When properly designed, HIC is an all‐aqueous, scalable, cost effective and predictable purification process. It can be implemented as a stand‐alone method or integrated into a dual‐column process alongside orthogonal techniques, such as AEX, to achieve even higher levels of product purity.