(M1130-06-40) Development and Validation of a Sensitive Hybridization-Electrochemiluminescence (H-ECL) Method for Quantitation of STK-002, an Antisense Oligonucleotide, in Human Serum

Purpose: STK-002 is an 18-mer antisense oligonucleotide (ASO) containing 2’-O-methoxyethyl (MOE) and phosphorothioate modifications and is administered via intravitreal injection (IVT). STK-002 is being developed for the treatment of autosomal dominant optic atrophy (ADOA) in patients aged 6 years and above. Systemic exposure of IVT drugs is generally low because of relatively low dose levels used via this administration route compared to systemic administration, therefore, requiring a sensitive bioanalytical assay to assess the pharmacokinetic profile of the drug. H-ECL platform has proven to be more sensitive than hybridization ELISAs (HELISA) for quantitating ASOs [1]. STK-002 quantitation in monkey serum achieved >10-fold sensitivity improvement with H-ECL method over HELISA, thus this method was selected for development in human serum that included assessments of two hybridization formats (i.e., single probe versus dual probes), and one-step versus multi-step hybridizations. The results from these evaluations and the validation data from the selected format will be presented.

Methods: In all assay formats tested during method development, STK-002 was annealed to a complementary, biotinylated capture probe and a complementary detection probe, which was conjugated to a ruthenium (Ru) tag; The capture and detection probes were custom synthesized and included 2’-O-methyl (2’-OMe) and locked nucleic acids modified ribonucleosides. The solution containing hybridized analyte and probes is plated onto a streptavidin coated MSD plate, after a blocking incubation. Following the analyte-probe incubation, MSD gold read buffer is added to the plate and then the ECL signal is read on the MESO QuickPlex SQ120 imager. One single probe method and three dual probe methods were tested. The single probe, complementary to STK-002, was biotinylated at the 3’-end and conjugated to Ru-tag at the 5’-end incorporating the components of the dual probes in a single sequence. In this method, micrococcal nuclease was added to cleave any unbound probes so only the hybridized probe and sample was detected. Using dual probes, three different sequences of hybridization were assessed. In method #1, the biotinylated capture probe, sample containing ASO, and the Ru-tagged detection probe were hybridized and then added to the streptavidin coated plate. In method #2, the biotinylated capture probe was coated onto the streptavidin plate. The sample containing ASO and the Ru-tagged detection probe were hybridized separately and then added to the coated plate. In method # 3, the biotinylated capture probe was hybridized to the sample containing ASO and coated onto the plate. The Ru-tagged detection probe was added later. Based on the performance of the assay, the best assay format was validated following bioanalytical method validation guidelines [2-3]. The validation included assessments of inter- and intra-batch accuracy and precision, hook effect, dilution linearity, selectivity (matrix interference), stability in frozen and thawed matrix and in whole blood, freeze/thaw stability, and robustness. The effects of hemolysis and lipemia on the accuracy and precision of the assay were also assessed.

Results: The single probe method produced low signal and showed poor accuracy; therefore, this format was not pursued further. Out of three formats that were evaluated using dual probe, method #1 (one-step) resulted in broader dynamic range, higher sensitivity, and better precision and accuracy than method #2 and #3. Therefore, method #1 was further optimized and validated. An assay calibration range was obtained from 0.0250 ng/mL to 200 ng/mL in human serum with the LLOQ and ULOQ at 0.0500 ng/mL and 200 ng/mL, respectively. Inter- and intra-batch accuracy was within 2.71% difference from theoretical for each QC concentration level. The inter- and intra-batch precision, as measured by percent coefficient of variance was ≤12.6%, and total error was ≤13.2%. No apparent hook effect was observed at concentrations up to 100,000 ng/mL. Dilution linearity was established for a QC sample at 40,000 ng/mL concentration and up to 320,000-fold dilution. Matrix effects (n=10 serum lots), and selectivity in lipemic and hemolyzed samples were evaluated, and acceptance criteria were met. Short-term stability (24 hours at room temperature), long-term stability (22 days at -80 °C), and freeze-thaw stability (5 cycles) were also established.

Conclusion: The performance of the single probe method was not suitable for its further development. Out of the three hybridization formats that were evaluated for dual probes, dual probe method #1 (the one-step H-ECL method) was determined to provide the most sensitive, accurate, and precise quantitation of STK-002 in human serum. The method’s dynamic range was 0.0500 ng/mL to 200 ng/mL, with acceptable stability and sensitivity for use in clinical trials.

References: 1. Norris DA, Post N, Yu RZ, Greenlee S, Wang Y. Bioanalysis considerations on the pharmacokinetic evaluation of antisense therapeutics. Bioanalysis 2019, 11, (21), 1909–1912.
2. FDA Guidance for Industry, Bioanalytical Method Validation Guidance for Industry. U.S. Department of Health and Human Services, Food and Drug Administration, 2018.
3. EMA Guideline on Bioanalytical Method Validation, Feb 2012.
4. Meso Scale Diagnostics Technical Resources Page on Electrochemiluminescence. https://www.mesoscale.com/en/technical_resources/our_technology/ecl . (accessed 2023-07-12).

Acknowledgements: The authors wish to acknowledge Kyle Griewisch for performing the validation experiments at PPD Laboratory Services, part of Thermo Fisher Scientific.

Figure 1: Electrochemiluminescence (ECL) Diagram [4]
Bound analytes recruit the detection probe from solution holding them near the electrode surface. A read buffer is added which provides the appropriate chemical environment for ECL. A voltage is applied to the plate electrodes causing the Ru-tag near the electrode to emit light through a series of reduction and oxidation reactions. The instrument measures the intensity of emitted light and provides a quantitative measure of analyte in the sample.

Figure 2: Single-Probe Hybridization-ECL Method
The ASO is hybridized to the single probe coated onto the plate. The nuclease cleaves any unhybridized probe and only ASO bound to the probe results in ECL signal.

Figure 3: Dual-Probe Hybridization ECL-Methods
a. Dual probe hybridization method #1 where the biotinylated capture probe (red), ASO, and Ru-tagged detection probe (blue) are hybridized in 1-step then added to the plate.
b. Dual probe hybridization method # 2 where the Ru-tagged detection probe (blue) and ASO are hybridized then added to the plate coated with the biotinylated capture probe (red) for a 2-step hybridization.
c. Dual probe hybridization method #3 where the Ru-tagged detection probe (blue) is added to the plate with hybridized biotinylated capture probe (red) and ASO for a 2-step hybridization.