Improved Recovery of a Lipid Conjugated Antisense Oligonucleotide from Human Plasma using the OligoWorks™ SPE Microplate Kit and an Optimized Protocol

Significance of the Topic

Oligonucleotide-based therapies are rapidly expanding in clinical development and approved applications, driven by the need for targeted gene modulation in genetic, neurological, cardiovascular, and oncological diseases.
Accurate bioanalytical quantification of these large molecules in complex matrices such as human plasma is essential for ADME/DMPK studies, yet is challenged by low circulating concentrations, strong protein binding, and the presence of lipid conjugates that reduce extraction recovery and sensitivity.

Objectives and Study Overview

The main objective was to enhance recovery and sensitivity for a lipid-conjugated antisense oligonucleotide (ASO) from human plasma using the OligoWorks™ SPE Microplate Kit combined with an optimized sample preparation protocol.
A 16-mer palmitate-conjugated ASO standard was spiked into 100 µL plasma samples and processed to assess extraction performance before and after protocol improvements.

Methodology

• Sample Pretreatment
  Plasma samples (100 µL) were spiked at 0.1 pmol/µL with the lipid-conjugated ASO and subjected to Proteinase K digestion using the RapiZyme module. Initial conditions were 1 hour at 55 °C; optimized conditions extended digestion to 2 hours, doubled Proteinase K concentration, and included 1% NP-40 Alternative surfactant to disrupt protein binding.
• SPE Purification
  Post-digestion plasma (180 µL) was applied to a 96-well mixed-mode WAX microplate employing OligoWorks sorbent. Washing steps were performed with adjusted methanol concentrations to improve lipid capture, and analytes were eluted in 25 µL of MS-compatible solvent without evaporation or reconstitution.
• Calibration and Quantification
  A calibration range of 5–2500 ng/mL in sodium heparin plasma was established. Multiple reaction monitoring (MRM) was used to evaluate linearity, sensitivity, and accuracy.

Instrumentation

• Waters ACQUITY I-Class PLUS UPLC System with ACQUITY Premier Oligonucleotide BEH C18 Column (1.7 µm, 2.1 × 50 mm)
• Waters Xevo TQ-XS Tandem Quadrupole Mass Spectrometer with negative electrospray ionization
• Alternative configuration: ACQUITY Premier LC System coupled to a Xevo TQ Absolute Mass Spectrometer for enhanced sensitivity

Key Results and Discussion

Initial recoveries across anticoagulants were low to moderate under the standard protocol. Optimization of digestion time, enzyme concentration, and surfactant addition yielded quantitative recovery (99%) of the lipid-conjugated ASO.
The method demonstrated excellent linearity (R² ≥ 0.99), a lower limit of quantification of 5 ng/mL, and accuracy within ±15% across all calibration points from only 100 µL plasma.

Benefits and Practical Applications

• Standardized kit-based workflow reduced method development time and simplified protocol optimization.
• Efficient use of small sample volumes (<100 µL) for low-abundance analytes.
• Automation-friendly microplate format enables high throughput and consistent performance.
• MS-compatible eluate eliminates the need for extract concentration steps.

Future Trends and Potential Applications

Ongoing advancements may include integration of microfluidic platforms, further refinements in mixed-mode sorbents for diverse oligonucleotide chemistries, and coupling with ultra-high-sensitivity mass spectrometers for sub-ng/mL quantification.
Expanding the workflow to additional biological matrices such as tissue homogenates and cerebrospinal fluid will support next-generation oligonucleotide therapeutics.

Conclusion

The optimized OligoWorks SPE Microplate Kit protocol achieves robust, near-complete recovery of lipid-conjugated ASOs from minimal plasma volumes, delivering high sensitivity and accuracy for bioanalytical applications in ADME/DMPK research.

References

• Transparency Market Research. Oligonucleotides Market Outlook 2034: Projected to Reach US$ 13.1 Billion by 2034, 2024.
• Lilly. Overview and Outlook for RNA-Based Therapies. Whitepaper, May 2024.
• Tran P, Weldemichael T, Liu Z, Li HY. Delivery of Oligonucleotides: Efficiency with Lipid Conjugation and Clinical Outcome. Pharmaceutics. 2022;14(2):342. doi:10.3390/pharmaceutics14020342.
• Araya M, Finny AS, Lee M, et al. Elements of Robust SPE-Based Oligonucleotide Extraction. Waters Application Note 720008414; 2023.