Lardeux Honorine, Bagci Selin, Gao Mimi, Holkenjans Wiebke, Pell Reinhard, Guillarme Davy
School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
Bayer AG, 42096 Wuppertal, Germany.
J Chromatogr A. 2025 Jan 4;1739:465523. doi: 10.1016/j.chroma.2024.465523. Epub 2024 Nov 14.
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.
离子对反相液相色谱法（IP-RPLC）显然被公认为分析治疗性寡核苷酸（ONs）的金标准。最近的研究表明，ONs在IP-RPLC中表现出开关保留行为，这意味着乙腈（ACN）比例的微小变化会显著影响保留。然而，这种行为最初仅在单一流动相条件下得到证明。本研究的目的是通过在广泛的流动相条件下测量S值（保留模型的斜率，log k对%ACN），更深入地了解ON的洗脱行为。我们系统地计算了20聚体和100聚体模型ON在各种条件下的S值，包括不同的离子对试剂、离子对浓度、流动相pH值、柱温以及两种不同的缓冲酸。我们证明，这些流动相条件对S值的影响顺序如下：离子对疏水性>离子对浓度>柱温>缓冲酸>流动相pH值。这种趋势的主要解释是，降低离子对保留机制的流动相条件（如低离子对疏水性或浓度）将增强%ACN对保留的影响，导致更高的S值。在研究的第二部分，利用这一知识为两种治疗性寡核苷酸开发了超快速分离方法：一种未进行硫代磷酸酯（PS）修饰的20聚体反义寡核苷酸（ASO）和一种包含某些PS修饰的大单向导RNA（sgRNA）。优化流动相条件以最大化S值，同时防止非对映异构体的分离。需要注意的是，要从使用超短柱中受益，S值至少需要达到30。这种方法可以使用5 mm长的色谱柱在不到一分钟的时间内成功分离主要成分（ASO和sgRNA）及相关杂质。
