(T1530-09-54) Sensitive Bioanalysis of Anti-sense Oligonucleotides through Mitigation of Non-specific Binding and Improved LC-MS/MS Performance

Purpose: Highly sensitive and specific LC-MS/MS and sample preparative methods were developed to routinely analyze antisense oligonucleotides (ranging from 18 to 33-mer) at sub ng/mL levels in human plasma. Oligonucleotide LC-MS/MS bioanalysis is challenging for a large number of reasons, including but not limited to: Electrospray negative ion performance; multiply charged nature of oligonucleotides; complex matrix background interference and protein removal; non-specific binding to sample preparative and LC system surfaces; and identifying robust and routine chromatographic methods1,2. This work addresses aspects of all these challenges in order to produce a sensitive and robust method suitable for routine analysis of antisense oligonucleotides.

Methods: Plasma samples were spiked at concentrations from 0.1 ng/mL to 10000 ng/mL with multiple antisense oligonucleotides containing 2’-MOE modified bases, GalNAc conjugate and/or phosphorothioate linkers (GEM91, Eluforsen, Fomivirsen, Mipomersen, Nusinersen and a GalNac conjugated Oligonucleotide). A simple 2-step LLE sample extraction was used for sample purification and concentration of 100 µl plasma samples. LC-MS/MS analysis of oligonucleotides was performed on a Waters™ TQ Absolute tandem quadrupole mass spectrometer using negative electrospray ionization (ESI-) and multiple-reaction monitoring mode (MRM). Chromatographic separation was performed using an ACQUITY™ Premier Oligonucleotide C18 Column and ACQUITY Premier System to increases analyte recovery and reproducibility by minimizing analyte/surface interactions. Prior to running samples, system checks were performed using readily available MassPrep OST standards to ensure system performance using a well characterized range of polythymidine oligonucleotide ladder standards (15-35mer). A flow rate of 0.5 mL/min and shallow gradient was employed using 100 mM hexafluoroisopropanol containing 15mM diisopropylethylamine in water or acetonitrile. Total analysis time was 5 minutes. Injection volumes of the extracted samples were 10-20 μL.

Results: Oligonucleotide MRM transitions were evaluated across a broad range of parent charge states and fragment masses for optimum performance.  Enhanced negative ion mode detection capabilities enabled improved counts, S/N and detection limits of the assay as compared to previous generation TQ XS instrumentation. Intra- and inter-day studies were performed, with QCs and LOQs being process using standard bioanalytical method validation criteria resulting in typical LLOQs of 0.1 ng/mL.

Conclusion: Data shows that routine sub ng/mL oligonucleotide quantitation of antisense oligonucleotides is possible using LC-MS/MS systems and meets criteria that is suitable for use in regulated and discovery bioanalytic settings. Improved oligonucleotide recovery and LC-MS/MS sensitivity was possible by mitigating non-specific binding on all surfaces that can interact with the biologics.

References: 1. Jennifer M Nguyen , Martin Gilar, Brooke Koshel, Michael Donegan, Jason MacLean, Zhimin Li & Matthew A Lauber, ‘Assessing the impact of nonspecific binding on oligonucleotide bioanalysis’, Future Science, BIOANALYSIS VOL. 13, NO. 16
2. Suma Veeramachineni, Mark D. Wrona, 'Sensitive LC-MS/MS Bioanalytical Quantitation of Antisense Oligonucleotides', Waters Corporation, Application Note, 2022.

Figure 1. Standard curve for GEM 91 analyzed on TQ Absolute showing 5x linear dynamic range, inset shows 0.1 to 10 ng/mL standards



Figure 2. Representative chromatogram showing 0.1 ng/mL standard (LLOQ) overlaid with matrix blank (green trace)