Comparison of multiple bioanalytical assay platforms for the quantitation of siRNA therapeutics
Creators: name: Agrawal, Karan
name: Calliste, Laurelle K
name: Ji, Shaofei
name: Xu, Shengsheng
name: Ayers, Stephen A
name: Jian, Wenying
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: <b>Aim:</b> Oligonucleotide therapeutics can be quantified using various bioanalytical methods, and these methods have been compared extensively. However, few comparisons exist where the same analyte is evaluated by multiple assay platforms. <b>Materials &amp; methods:</b> Hybrid LC-MS, SPE-LC-MS, HELISA and SL-RT-qPCR methods were developed for an siRNA analyte, and samples from a pharmacokinetic study were analyzed by all four methods. <b>Results:</b> All assay platforms provided comparable data, though higher concentrations were observed using the non-LC-MS assays. Hybrid LC-MS and SL-RT-qPCR were the most sensitive methodologies, and SL-RT-qPCR and HELISA demonstrated the highest throughput. <b>Conclusion:</b> Each assay platform is suitable for oligonucleotide bioanalysis, and the ultimate choice of methodology will depend on the prioritization of needs such as sensitivity, specificity and throughput. Oligonucleotide therapeutics can be quantified using various bioanalytical methods, with LC-MS and hybridization ELISA (HELISA) approaches commonly used to support regulatory bioanalytical submissions. Several comparisons between oligonucleotide bioanalytical assays have been published, but few evaluate the same analyte by multiple assay platforms. Therefore, it is difficult to identify the extent to which assays differ due to factors inherent to the assay platform versus factors inherent to the compound being analyzed. To better understand the strengths and limitations of common oligonucleotide assay platforms, hybrid LC-MS, solid phase extraction-LC-MS (SPE-LC-MS), 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 evaluated provided comparable data for the study samples, though HELISA and SL-RT-qPCR tended to generate higher observed concentrations relative to the LC-MS assays, possibly due to quantification of both the parent analyte and its metabolites, which indicates a lack of specificity. Hybrid LC-MS and SL-RT-qPCR demonstrated the highest sensitivity, and SL-RT-qPCR and HELISA demonstrated the highest throughput. Evaluation of assay performances indicate that all assay formats could generally be validated to standards necessary to support regulatory bioanalytical submissions. 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.