Development and validation of LC-MS-based bioanalytical methods for oligonucleotide therapeutics: points to consider.

The analysis of oligonucleotide therapeutics, including antisense oligonucleotides and siRNAs, using existing bioanalytical methods presents unique analytical challenges owing to their distinctive physicochemical properties. Although the ICH M10 guideline provides general bioanalytical validation frameworks, specific considerations for oligonucleotide therapeutics remain undefined, highlighting an urgent need for targeted points to be considered. This white paper presents comprehensive recommendations developed through expert consensus and a literature review by the Japan Agency for Medical Research and Development research groups, addressing the critical technical challenges in liquid chromatography-mass spectrometry (LC-MS)-based bioanalytical methods for oligonucleotide therapeutics. It focuses on ion-pair reversed-phase LC-MS methodologies and emerging techniques, such as hybridization extraction, addressing key issues, including mobile phase stability and degradation, metal adsorption mitigation, carryover prevention, internal standard selection, and matrix effects unique to nucleic acids. The comprehensive coverage includes sample preparation strategies, chromatographic optimization, mass spectrometric detection approaches, and validation parameters adapted from ICH M10, with fit-for-purpose modifications on a case-by-case basis when conventional acceptance criteria cannot be applied. This white paper provides practical recommendations for the development and validation of analytical methods specific to oligonucleotide therapeutics, supports regulatory submissions, and promotes the international harmonization of bioanalytical standards in this rapidly expanding therapeutic modality.