(T1030-06-37) Strategies for Measuring Oligonucleotides Using a Fully Automated Microfluidic Immunoassay System

Purpose: The rapidly growing field of oligonucleotide medicine provides unique opportunities to treat genetic diseases and provide new treatments and vaccines for viral diseases. As of March 2023, there are 18 FDA approved oligonucleotide-based drugs (excluding vaccines), and many more are currently in clinical trials. Strategies for pharmacokinetic (PK) and pharmacodynamic (PD) measurements of these drugs, which are typically structural variants of DNA or RNA to increase pairing and biologic stability, often utilizes the base pairing capabilities in hybridization assays which mimic an ELISA format. However, these methods are often time and labor intensive. It was previously shown these assays could be transferred to an automated microfluidic format (1). In this study, three automated assay formats, dual hybridization assay (DHA) and nuclease cutting assay (NCA) using S1 nuclease or Micrococcyl nuclease, were further developed to compare assay performance and detection of metabolites.

Methods: The DHA and the NCA formats (Micrococcal and S1 nuclease) were run on the Gyrolab automated microfluidic immunoassay platform. The DHA utilizes one capture oligo and one detection oligo and an incubation step implementing the Mixing CD. If not held in place by the analyte, the detection oligo is washed away. The NCA utilizes a full-length oligo probe biotinylated in one end and with a conjugated fluorophore on the other end. Single stranded probes that are unbound or partially hybridized are cleaved by nuclease and the detection molecule is washed away. The analyte for both assays mimicked the anti-sense oligonucleotide drug casimersen (Amondys 45™). Metabolite analogs for 5’ and 3’ n-1, n-2, and n-3 degradation products were also synthesized and tested for cross-reactivity using both the NCA and DHA.

Results: Assays quantifying the oligo therapeutic analog of casimersen were successfully automated on the Gyrolab microfluidic platform using two formats, DHA and NCA, with two different nucleases in the NCA assay (Micrococcal nuclease and S1 nuclease). For the DHA, the assay range was 0.64 – 10,000 pM and the LLOQ 2 pM. For both the S1 and Micrococcal NCAs the assay range was approximately 3 – 20,000 pM and the LLOQ estimated at 10 pM. Cross-reactivity for the DHA at n-1 for the 5’ metabolite was significant, at approximately 40%. No significant cross reactivity was observed for N-1 at the 3’ end, or the n-2 and n-3 metabolites at either the 5’ or the 3’ ends. There was some cross-reactivity at the 3’ end for the NCA, at approximately 30% for n-1, n-2, and n-3 metabolites.

Conclusion: The dynamic range of the three assays was acceptable, indicating all three methods are viable approaches to the quantification of oligo therapeutics in biological matrices using the automated microfluidic system. The assay runtime of about 2.5 hours of which the vast majority is automated represented a significant savings of hands-on and total assay time. Cross-reactivity for the DHA format was lower than expected and may be a reflection of the robustnessness of the microfluidic Gyrolab assay format or the oligo analog sequence modifications. There is potential to reduce cross-reactivity further for the NCA methods by adding a nuclease wash step to the assay.

References: 1. James Sharkey, John Chappell, and Frida Löthberg. Strategies for measuring oligonucleotides using a fully automated microfluidic immunoassay system, poster presentation at AAPS NBC 2023.

Dual hybridization and nuclease cutting assay formats for the automated microfluidic assay

Assay performance for DHA and NCA oligo assay formats

Cross-reactivity of metabolites for the micrococcal nuclease cutting assay