ELEMENTS OF ROBUST MODIFIED OLIGONUCLEOTIDE EXTRACTION THROUGH SPE

Significance of the Topic

The extraction and quantification of therapeutic oligonucleotides in biological matrices are essential for reliable drug metabolism and pharmacokinetic (DMPK) analyses. Efficient purification reduces matrix effects, enhances sensitivity, and supports the development of oligonucleotide-based therapies.

Objectives and Study Overview

This study aimed to develop a robust solid-phase extraction (SPE) workflow for both unmodified and lipid-conjugated oligonucleotides using a tailored ion-exchange sorbent. Key goals included screening sorbent batches, optimizing sample preparation with proteinase digestion, and demonstrating reproducible recovery across analytes.

Methodology

• Sample Preparation: Rat plasma was denatured and proteins digested with a proteinase K module under denaturing conditions to release bound oligonucleotides.
• SPE Sorbent Screening: Mixed-mode weak anion-exchange sorbent batches (Oasis WAX) were evaluated in batch testing with a 20-mer ssDNA standard and a lipid-conjugated antisense oligonucleotide (ASO).
• Chromatographic Analysis: IP-RP-LC-UV assays on ACQUITY UPLC with BEH C18 column monitored at 260 nm. IP-RP-LC-UV-MS profiling of lipid-ASO was performed on a BioAccord system using HFIP/DIPEA buffers. SEC assessed digestion completeness of plasma proteins.

Used Instrumentation

• Acquity UPLC with BEH C18 column (1.7 µm, 2.1×50 mm) for IP-RP-LC-UV
• BioAccord™ System with RDa detector for IP-RP-LC-UV-MS
• ACQUITY Premier SEC column (4.6×150 mm) for protein digestion monitoring
• OligoWorks™ SPE kit cartridges packed with selected Oasis WAX sorbent

Main Results and Discussion

• Selected Oasis WAX batches delivered high and consistent recovery for both ssDNA and lipid-ASO standards.
• UV chromatograms confirmed efficient removal of interferences and clean elution profiles after SPE.
• Lipid-conjugated ASO required elevated organic content for optimal LC-MS separation; two diastereomer peaks were partially resolved.
• Proteinase K digestion achieved complete plasma protein breakdown within 40 minutes, as shown by SEC profiles.

Benefits and Practical Applications

The optimized SPE protocol enables reproducible extraction of diverse oligonucleotide chemistries, supporting bioanalytical workflows in drug development and quality control. Certified sorbent performance ensures batch-to-batch consistency, and standard oligo references facilitate method validation.

Future Trends and Applications

• Expansion to emerging oligonucleotide modalities (e.g., siRNA, gapmers, PMOs).
• Integration with high-resolution mass spectrometry for detailed metabolite profiling.
• Automation of SPE workflows and miniaturization for high-throughput screening.
• Development of next-generation mixed-mode sorbents to improve selectivity and capacity.

Conclusion

The OligoWorks SPE kit, leveraging precisely controlled Oasis WAX sorbent batches, provides a robust, reproducible solution for extracting modified and unmodified oligonucleotides from complex biological samples. This workflow enhances bioanalytical accuracy and supports drug discovery efforts.

References

• Waters Corporation. OligoWorks SPE Kit information. www.waters.com/oligoworks