(T1330-02-11) Oligonucleotide Dual Hybridization Assay Using Offline Pre-incubation followed by an Automated Microfluidic Method

Purpose: The development of oligo-based therapeutics is a rapidly growing effort, with opportunities for treatment of orphan and genetic diseases. Currently there is a need for improved bioanalysis methods for the detection of oligo therapies for pharmacokinetic (PK) and pharmacodynamic (PD) analysis. In this feasibility study, the performance of a hybridization ligand-binding assay for the detection of the anti-sense oligonucleotide (ASO) drug casimersen (Amondys 45™) for the treatment of Duchenne muscular dystrophy (DMD) using a labeled oligonucleotide probe with offline incubation followed by an automated microfluidic method was evaluated. While the oligo assay in a fully automated format using the Gyrolab^® platform and Mixing CD 96 has been previously presented,¹ this study investigates the use of pre-incubation of sample and capture probe to offer greater flexibility in assay format and detection range based on the choice of CD and requirements of the study.

Methods: Capture and detection probes, and analyte were added together to wells of a microplate and pre-incubated at RT for 1 hour. Capture reagent was a biotinylated oligo probe complementary to the analyte, which mimicked the anti-sense oligonucleotide drug casimersen (Amondys 45™). Detection reagent was an oligo probe complementary to the sample labeled with Alexa Fluor 647 Samples of analyte mimicking biological samples were prepared in human serum then diluted 1:2. In the automated method, pre-incubated samples with capture and detection probe were loaded onto the CD and bound to the streptavidin-coated bead in the flow-through column of the Gyrolab Bioaffy 1000 CD. Unbound detection oligo was washed away, then the complex was detected automatically laser-induced fluorescence (LIF) of the flow-through columns (Figure 1).
Analyte Sequence
Based on AMONDY 45 phosphorodiamidate morpholino oligomer (PMO) synthesized to match the published base sequence².
5´ CAA TGC CAT CCT GGA GTT CCT G 3´
Capture Reagent Sequence
5´ GGA TGG CAT TG 3´-Biotin
Detection Reagent
Alexa Fluor^® 647-5´ CAG GAA CTC CA 3’

Results: Precision of the assay run 3x by 3 different operators was excellent, with %CV under 12% for concentrations from 5 to 8000 pM, and relative error below 20% (Table 1, Figure 1). Dilutional linearity for samples at 5000 (1/10), 500 1/100), 50 (1/1000), and 5 (1/10,000), was at 100%, 94.7%, 94.6%, and 100.8% of expected values, respectively (Figure 3). Dilutional linearity data demonstrates no evidence of matrix effects from human serum. The time to complete the incubation with automated assay was [approximately 2 hours to complete the assay in total, including the 1 hour pre-incubation. The data presented here using an offline incubation of the sample and the complementary probes before running them on the Gyrolab system allows flexibility in assay dynamic range by choice of Gyrolab Bioaffy CD. For high sensitivity requirements, the 4000 or 1000 nL CD can be selected. Alternatively, lower sensitivity and larger upper range assay detection could be achieved with the 20 or 200 nL CD. Offline incubation also allows for different incubation temperatures which may be particularly relevant dependent on the optimal temperature for hybridization or requirement of a heat treatment cycle.

Conclusion: The combination of offline incubation of the sample and capture probe followed by the automated microfluidic method including laser-induced fluorescence detection was effective in generating robust data with minimal labor and in a short amount of time. The precision and dilution linearity values demonstrated a robust assay and were in line with requirements for use in preclinical or clinical studies. While this was a feasibility study, this data supports further development of this assay.

References: 1. AMONDYS 45. Prescribing Information. Sarepta Therapeutics; 2021. https://www.amondys45.com/pi, accessed January 22, 2024.
2. Chappell, J. (2023, April 23-26). Strategies for measuring oligonucleotides using a fully automated microfluidic immunoassay system. [Poster presentation]. AAPS NBC, Philadelphia, PA, USA

Diagram of microfluidic oligonucleotide dual hybridization assay. The method is based on a 15 nL flow through affinity column built into microstructures on a microfluidic CD for automated immunoassay fluid flow and detection. In this assay, capture and detect probe along with oligonucleotide therapeutic sample were pre-incubated offline then loaded onto the microfluidic instrument where the complex was captured on the flow through column and unbound detection probe washed away.

Samples with therapeutic oligo in 1:1 human serum at concentrations from 5 to 8000 pM were run 3 operators in 3 separate runs.

Dilutional linearity for samples at 5000 (1/10), 500 1/100), 50 (1/1000), and 5 (1/10,000) was run by 3 operators in 3 separate runs.