Understanding the fundamentals of the on-off retention mechanism of oligonucleotides and their application to high throughput analysis

Ion-pair reversed-phase liquid chromatography (IP-RPLC) is clearly recognized as the gold standard for analyzing therapeutic oligonucleotides (ONs). Recent studies have shown that ONs exhibit an on-off retention behavior in IP-RPLC, meaning that minor changes in acetonitrile (ACN) proportion can significantly impact retention. However, this behavior was initially demonstrated with only a single mobile phase condition. The aim of this study is to gain a deeper understanding of ON elution behavior by measuring the S values (slope of the retention model, log k vs.%ACN) across a broad range of mobile phase conditions. We systematically calculated the S values for both a 20-mer and 100-mer model ON under various conditions, including different IP reagents, IP concentrations, mobile phase pH, column temperatures, and two different buffering acids. We demonstrated that these mobile phase conditions impact the S values in the following order: IP hydrophobicity > IP concentration > column temperature > buffering acid > mobile phase pH. The main explanation for this trend is that mobile phase conditions that reduce the ion-pair retention mechanism (such as low IP hydrophobicity or concentration) will enhance the impact of% ACN on retention, leading to higher S values. In the second part of the study, this knowledge was used to develop ultra-fast separations for two therapeutic oligonucleotides: a 20-mer antisense oligonucleotide (ASO) without phosphorothioate (PS) modifications and a large single guide RNA (sgRNA) that includes certain PS modifications. The mobile phase conditions were optimized to maximize S values, while preventing the separation of diastereomers. It is important to notice that an S-value of at least 30 is required to benefit from the use of ultra-short columns. This approach allows the successful separation of the main species (ASO and sgRNA) and related impurities in less than one minute using a 5 mm length column.