Comparison of Four Bioanalytical Assay Platforms for siRNA Therapeutic Quantitation

Oligonucleotide therapeutics can be quantified using various bioanalytical methods, and while these methods have been compared extensively, few comparisons exist where the same analyte is evaluated by multiple assay platforms. To better evaluate each assay platform, hybrid LC-MS, solid phase extraction-LC-MS (SPE-LC-MS), hybridization ELISA (HELISA), and stem loop-reverse transcription-quantitative PCR (SL-RT-qPCR) workflows were developed for an siRNA analyte, and samples from a pharmacokinetic study were analyzed by all four methods. All assay platforms provided comparable data for the study samples, though the non-LC-MS assays tended to generate higher observed concentrations relative to the LC-MS assays, suggesting a lack of specificity. Additionally, hybrid LC-MS and SL-RT-qPCR demonstrated the highest sensitivity, and SL-RT-qPCR and HELISA demonstrated the highest throughput. Therefore, it appears that each assay platform evaluated is suitable for oligonucleotide bioanalysis, and the ultimate choice of methodology will depend on which factors among sensitivity, specificity and throughput need to be prioritized.