Impurity profiling of lipid-conjugated oligonucleotides using reversed-phase with and without ion-pair reagents and hydrophilic interaction liquid chromatography.

Lipid-oligonucleotide conjugates (LONs) represent a next-generation class of biopharmaceuticals that couple nucleic-acid specificity with lipid-mediated delivery, enabling superior membrane affinity and cellular uptake, for highly targeted therapeutic action. Lipid type and conjugation position strongly influence hydrophobicity, retention, and interaction strength from the exposure of the lipid moiety, thereby affecting the chromatographic behavior of impurities such as shortmers and diastereomers. In this study, we systematically investigated the performance of different chromatographic modes, reversed-phase LC (with and without ion-pairing agents) and hydrophilic interaction LC (HILIC), and assessed the influence of mobile-phase additives on the retention behavior of both single- and double-stranded LONs. RPLC and IP-RPLC proved superior to HILIC for impurity profiling, offering higher separation resolution and MS compatibility. In contrast to naked ONs, LON retention did not follow an on-off mechanism in (IP-)RPLC mode. Moreover, LONs displayed the opposite retention order. A novel metric was developed to account for the hydrophobic contributions of both the oligonucleotide backbone and lipid conjugation, demonstrating a strong correlation with experimentally observed retention behavior in RPLC. High resolving power was achieved for diastereomeric impurity profiling of internally lipid-modified oligonucleotide (iLON) duplexes using a C4 column in RPLC mode, revealing that the extent of phosphorothioate modification suppression depends strongly on both the lipid type and its conjugation position. The resolving power of RPLC was demonstrated by separating >30 of the theoretical 64 diastereomers present in a siRNA sample.