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.