(M1030-06-35) Development and Optimization of a Highly Sensitive and Selective Anion Exchange Liquid Chromatography with Fluorescence Detection Assay for Quantitation of siRNA Oligonucleotide Therapeutic Compounds

Purpose: Determining the pharmacokinetics and biodistribution of a potential drug are key required assessments for all pharmaceutical compounds (1). A robust, accurate, and efficient bioanalytical method is essential for obtaining these data. Development of bioanalytical assays has been particularly complex for oligonucleotide therapeutics. Historically, the bioanalysis of oligonucleotides has been performed using immunoassays, liquid chromatographic assays, and quantitative polymerase chain reaction (qPCR) (2). Analytical techniques for small interfering RNA (siRNA) oligonucleotides have posed an additional challenge of a duplex of two RNA strands annealed through Watson-Crick base paired hydrogen bonds, which typically require extensive sample processing for analysis. Liquid chromatographic mass spectrometry (LC-MS) assays have been commonly used for analysis of siRNA oligonucleotides (3). Immunoassays and qPCR have demonstrated increased sensitivity compared to LC-MS assays but lack the ability to differentiate some metabolites from parent compounds (2, 4-5). We report here an anion exchange liquid chromatography with fluorescence detection (AEX-LC-FL) assay that provides increased sensitivity compared to traditional LC-MS methodologies in addition to increased compound selectivity when compared to immunoassays and qPCR.

Methods: A method was developed for the bioanalysis of Arrowhead Pharmaceuticals’ compound ADS-018, a siRNA conjugated to a lipid moiety at the 5’ terminus of the sense strand to improve activity and distribution of the oligonucleotide. Analysis of various biological matrices was performed using an AEX-LC-FL assay that allowed for detection of the antisense strand in samples by annealing to a complementary, fluorescence-labeled peptide nucleic acid (PNA) probe (atto-probe). Homogenized tissue, plasma, cerebrospinal fluid (CSF), and urine samples were prepared by spiking blank matrix with working solutions containing ADS-018. Spiked samples were first digested in sample buffer containing proteinase K for 30 minutes at 65 °C. The digested samples were then heated to 95 °C in the presence of PNA atto-probe for 15 minutes, then at 55 °C for 15 minutes, and then rapidly cooled to 4 °C to form atto-probe: antisense strand duplexes. Anion exchange chromatography with fluorescence detection was used to separate the atto-probe: antisense strand complexes from the excess of atto-probe reagent and chromatographic interferences. A fluorescence detector was used to detect the annealed atto-probe: antisense strand complexes for indirect quantitation of ADS-018.

Results: An AEX-LC-FL assay was developed and qualified for analysis of the siRNA conjugate, ADS-018, in various biological matrices with an analytical range of 0.250 -1000 ng/mL. Assay linearity and sensitivity were evaluated using two sample buffers. Accuracy and precision were evaluated and found to be acceptable across six concentrations in monkey brain homogenate (table 1). Matrix parallelism and assay performance were successfully evaluated in five different monkey matrices (brain homogenate, gastrocnemius homogenate, CSF, urine, and plasma) and sample buffer (matrix free) (table 2).

Conclusion: Previous research has focused on optimizing the PNA probe design for development of LC-FL assays (6). The selection of an appropriate probe is still critical to the development of an AEX-LC-FL assay. However, we have improved the specificity and sensitivity of our assay by focusing development efforts on selecting an optimal sample buffer. By optimizing the sample buffer, we were able to reduce chromatographic background, increase sensitivity, and increase linearity of the assay (figure 1). The developed AEX-LC-FL assay was highly sensitive and selective for the analysis of ADS-018 with an analytical range of 0.250 – 1000 ng/mL. The assay was free of most matrix-based influences and was easily applied for analysis across a broad range of biological matrices and tissue homogenates. These novel assay modifications show a significant benefit of AEX-LC-FL when compared to traditional LC-MS assays for biodistribution studies. The AEX-LC-FL assay improvements also demonstrated increased sensitivity (2 to 20-fold) when compared to LC-MS assays (4). Additionally, this new assay can separate truncated metabolites from full-length parent strands, which is an advantage over immunoassays or qPCR. Finally, the newly developed assay demonstrated broad application to the entire class of oligonucleotides with various conjugated designs, across multiple biological matrices, when used in the evaluation of PK and biodistribution for development of potential siRNA therapeutics.

References: 1. Geary, R.S., Norris, D., You, R., Bennett, C.F., Pharmacokinetics, biodistribution, and cell uptake of antisense oligonucleotides. Advanced Drug Delivery Reviews 87, 46-51 (2015).
2. Tremblay, G.A., Oldfield, P.R. Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues. Bioanalysis 1 (3), 595-609 (2009).
3. Xing, J., Arya, V., Reynolds, K.S., Rogers, H., Clinical pharmacology of RNA interference-based therapeutics: A summary based on food and drug administration-approve small interfering RNAs. Drug Metab Dispos 51, 193-198 (2023).
4. Agrawal, K., Calliste, L.K., Ji S., Xu, S, Ayers, S.A, Jian, W., Comparison of multiple bioanalytical assay platforms for the quantitation of siRNA therapeutics. Bioanalysis, DOI: 10.1080/17576180.2024.2350266.
5. Yeung, B.Z, Lu, Z., Wientjes, G.M., Au J.L.S, High sensitivity RT-qPCR assay of nonlabelled siRNA in small blood volume for pharmacokinetic studies: application to surviving siRNA. The AAPS Journal 17 (6), 1475-1482 (2015).
6. Tian, Q., Rogness, J., Meng, M., Li, Z., Quantitative determination of a siRNA (AD00370) in rat plasma using peptide nucleic acid probe and HPLC with fluorescence detection. Bioanalysis 9 (11), 861-872 (2017).

Acknowledgements: Funding for this research was provided by Arrowhead Pharmaceuticals, Inc. B. Krull and C. Cousineau are employees of Arrowhead Pharmaceuticals, Inc. and hold stock in Arrowhead Pharmaceuticals, Inc. No writing assistance was utilized in the production of this poster.
Blank monkey tissues used to prepare tissue homogenates in this research were collected as a terminal collection in Labcorp study 8480457 and supported by IACUC 078MF32-JUS15MAR2021.
The authors would like to thank Jeff Prusakiewicz, Ryan Sheeler, Nagsen Gautam, Washim Khan, and Jack Shi for reviewing this poster.

Intra-assay accuracy and precision results from samples prepared in monkey brain homogenate.

Matrix parallelism results and quality control statistics across multiple matrices.

Comparison of conventional assay chromatography and improved assay chromatography.