Identification of oligonucleotide drug impurities using heart-cutting two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS)

Background Oligonucleotide-based drugs have gained significant attention as a promising class of therapeutics due to their precise ability to regulate gene expression. However, the presence of impurities in these drugs can compromise their safety, efficacy, and stability. Therefore, the identification and detailed analysis of these impurities are crucial. Traditional methods may struggle to detect subtle impurities, such as oxidation and hydrolysis products, which necessitate the development of more advanced and reliable analytical techniques for oligonucleotide therapeutics. Results In this study, we introduce a novel analytical method that combines heart-cutting two-dimensional liquid chromatography (2D-LC) with tandem mass spectrometry (MS/MS) to effectively separate, analyze, and identify impurities in oligonucleotide-based drugs. The first dimension utilizes anion exchange chromatography (AEX) to separate oligonucleotides based on their negative charge, while the second dimension employs reverse ion-pair chromatography (RIPC) for further purification and compatibility with mass spectrometry. This 2D-LC-MS/MS approach provides a sensitive and accurate means of identifying and quantifying impurities, including oxidation and hydrolysis products. The method was applied to two RNA interference (RNAi) drugs, Givosiran and Patisiran, where 3 and 20 impurities were identified, respectively. Additionally, sequencing analysis using data-dependent acquisition (DDA-MS/MS) enabled the determination of the molecular weight and structural characteristics of these impurities, offering a comprehensive and detailed view of the impurity profiles. Significance This novel heart-cutting 2D-LC-MS/MS method offers a significant advancement in the analysis of impurities in oligonucleotide-based therapeutics. It provides high sensitivity, enabling the identification of subtle impurities that are often challenging to detect. By applying this technique to RNAi drugs, we demonstrate its potential to enhance the safety, efficacy, and stability of oligonucleotide-based therapies, making it a valuable tool for the pharmaceutical industry.