(M1130-01-06) Challenges and Solutions in Ion-Pair Reversed-Phase LC–MS/MS Assays for the Quantitation of Novel Oligonucleotides: A Case Study

Purpose: Oligonucleotide therapeutics (OT), including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), enable precise modulation of gene expression at the nucleic acid level, thereby facilitating the rapid targeting of previously undruggable pathways. They offer high specificity, tunable pharmacokinetics, and minimal off-target effects. Compared to ASOs and siRNAs, aptamers are less widely adopted but stand out as single-stranded nucleic acids that fold into defined three-dimensional conformations, enabling antibody-like target binding while being produced synthetically, reducing immunogenicity and streamlining manufacturing. BB-031 is a first-in-class RNA aptamer designed to target von Willebrand Factor (vWF), a critical structural component in the blood clotting cascade involved in thrombus formation, stabilization, and expansion. Preclinical studies have demonstrated that BB-031 outperforms recombinant tissue plasminogen activator (rTPA) in ischemic stroke models by reopening blocked arteries with enhanced safety and reduced bleeding risks. However, BB-031’s thrombolytic effect can be neutralized when BB-025 is administered after BB-031, forming a stable complex. In this study, we aimed to develop and validate assays to quantify both BB-031 and BB-025 in human plasma to support clinical studies. A systematic in-house method development procedure was applied featuring the following novel aspects: 1. Method Development Procedure (MDP): Leveraging many years of experience in oligonucleotide assays and decades of LC–MS/MS bioanalysis expertise, we have established a standardized MDP with multiple protocols ensuring all conditions were comprehensively evaluated. 2. Selection of internal standard (IS): Initially, a stable isotope-labeled internal standard (SIL-IS) was synthesized but could not be used due to impurity issues. As an alternative, oligo IS candidates from our internal IS library were screened. An IS with similar chromatographic properties was selected, and ultimately worked well during validation. 3. Robustness Improvement: Method robustness was enhanced by using column backflushing and solid phase extraction (SPE) centrifugation. A third pump allows the column to be flushed after each injection, extending the HPLC column’s lifespan and maintaining performance even after thousands of injections. 4. Prevention of Non-Specific Binding (NSB): To determine solvents that prevent non-specific binding in stock solutions, working solutions, and sample extract reconstitution, the MDP non-specific binding protocol was employed. The most effective conditions were identified and verified under stressed conditions.

Methods: A 25-µL of plasma sample was spiked with an analog IS and extracted by weak cationic exchange SPE using a mixture of ammonium hydroxide, ammonium bicarbonate, tetrahydrofuran, and acetonitrile (MeCN), and water as elution solvent. The elute was dried down with nitrogen gas and reconstituted with a mixture of hexafluoroisopropanol (HFIP), diisopropylethylamine (DIPEA), Ethylenediaminetetraacetic acid (EDTA), and water. Extracts were then injected into a Thermo DNAPac RP column with the mobile phases composed of HFIP, DIPEA, EDTA, MeCN, and water. A Sciex Triple Quad 6500+ MS/MS system was used in negative electrospray ionization mode to monitor BB-025, BB-031, and the IS at ion transitions of 807.3→287.1, 787.3→287.1, and 820.4→134.2 respectively.

Results: At the early stage of method development, we encountered several challenges, including NSB, IS selection, and a short column lifetime. To address these issues, we applied a standardized MDP comprising nine protocols designed to identify and resolve potential problems such as NSB, low recovery, solubility, and stability. Since BB-025 and BB-031 are highly hydrophilic and prone to NSB, we evaluated various additives—including Triton X-100, CHAPS, EDTA, and NaCl—and found that saline effectively prevented NSB. Initially, we evaluated a synthesized SIL-IS, BB-031-d27, but significant impurities and analyte interference were observed. Consequently, we screened 10 IS candidates from our oligonucleotide IS library and identified one candidate that provided comparable chromatographic and retention properties, demonstrating its suitability for tracking sample extraction and instrumentation variations. The LC gradient was optimized to minimize baseline noise by screening different columns and mobile phases according to the MDP. In addition to the binary pump, a third pump was incorporated to enable column backflushing after each injection, significantly prolonging the HPLC column’s lifespan and maintaining optimal performance even after thousands of injections. We also screened SPE elution solvents with varying percentages of methanol and found that a water/MeCN (60/40, v/v) solution effectively removed salts and interferences while delivering consistent results. Rather than using a traditional positive pressure manifold, we streamlined the extraction process by employing centrifugation, completing the entire extraction in approximately 30 minutes. After thorough method development, the method was validated over a concentration range of 50.0 to 25,000 ng/mL, with a dilution quality control (DQC) set at 300,000 ng/mL. Inter-assay accuracy ranged from –2.7% to 7.5%, and inter-assay precision ranged from 5.4% to 10.9%. Specificity, selectivity, matrix effects, and stability were evaluated according to M10 guidance and met the recommended criteria.

Conclusion: We have successfully developed and validated a robust LC-MS/MS assay to quantify BB-031 and BB-025 in human plasma. Systematic optimization resolved issues with non-specific binding, recovery, and column lifespan, achieving excellent sensitivity and accuracy. In summary, it is the first published combination assay for BB-031 and BB-025 in human plasma.


Typical Chromatogram (Eluted in the Order of BB-025, IS, and BB-031)


Precision and Accuracy of BB-025


Typical Curve of BB-031