Automated and systematic screening of ion-pair reversed-phase LC conditions accelerating assay development of oligonucleotides in pharmaceutical mixtures.

The pharmaceutical industry is rapidly advancing toward new drug modalities to address unmet medical needs, requiring the development of advanced analytical strategies for deployment of effective and reliable assays. Ion-pair reversed-phase liquid chromatography (IP-RPLC) is widely regarded as a reference technique for the analysis of therapeutic oligonucleotides; however, method development remains challenging because retention and selectivity are highly sensitive to the nature of the ion-pairing reagent, organic modifier, and stationary phase. Here, we present a high-throughput IP-RPLC screening platform to systematically evaluate these parameters for the separation of challenging oligonucleotide impurities. The setup combined three bioinert reversed-phase columns of different chemistries (i.e., BEH C18, BEH phenyl, and BEH C18 Shield) with multiple mobile phase compositions containing alkylamines of increasing hydrophobicity with either methanol or acetonitrile as organic modifier. Three representative commercially available oligonucleotide sample sets were investigated: shortmers and longmers from a synthetic antisense oligonucleotide, phosphodiester/phosphorothioate (PO/PS) oxidation variants related to 21-mer fomivirsen, and low-purity 18-mer synthetic ASO samples containing a broad range of process-related impurities. Separation quality was evaluated through the Separation Quality Factor (SQF). For shortmers/longmers separation, 20 mM butylamine with methanol on a phenyl column provided the best overall compromise in terms of resolution and number of detected oligonucleotide peaks, enabling baseline separation of five species in <5 min after gradient optimization. For the PO/PS impurity series, the screening highlighted the strong influence of backbone chemistry on retention and demonstrated that PS content differences could be effectively discriminated using 25 mM triethylamine with methanol on a phenyl column. Finally, impurity profiling of low-purity synthetic ASO showed that 1 mM hexylamine with acetonitrile on a BEH C18 Shield column provided the most informative chromatographic fingerprint, revealing increasingly complex impurity patterns as sample purity decreased from > 95% to 30%. Overall, the proposed automated screening strategy enabled rapid identification of suitable IP-RPLC conditions for distinct oligonucleotide impurity classes and appears particularly well suited for accelerating method development for emerging therapeutic oligonucleotides.