High-sensitivity quantification of antisense oligonucleotides for pharmacokinetic characterization

Aim: Antisense Oligonucleotides (ASOs) are a fast-growing drug modality. Pharmacokinetic characterization and accurate quantification of ASOs is critical for drug development. LC-MS and hybridization immunoassays are common methods to quantify ASOs but may lack sensitivity. In this study we aimed to develop an ASO quantification method with improved sensitivity. Methods: We developed a branched DNA approach for ASO quantification and compared it with hybridization immunoassays. Results: The branched DNA assay showed significantly improved sensitivity, with LLOQ 31.25 pg/ml in plasma, 6.4-and 16-fold higher than dual-probe hybridization electrochemiluminescence and single-probe hybridization ELISA, respectively, with adequate precision, accuracy, selectivity and specificity and acceptable matrix interference. Conclusion: Branched DNA for ASO quantification has significantly higher sensitivity and lower hemolysis interference.

antisense oligonucleotides (ASOs); bioanalysis; branched DNA (bDNA); dual-probe hybridization immunoassay (H-ECL); high-sensitivity quantification; single-probe hybridization nuclease protection assay (H-ELISA).