Optimizing Separation of Oligonucleotides with Anion‑Exchange Chromatography

Significance of Oligonucleotide Purification

Effective purification of synthetic oligonucleotides is essential for their use in therapeutics, gene editing, and molecular diagnostics. Impurities such as truncated sequences, base loss, and variable adducts can affect biological activity, quantitation accuracy, and regulatory compliance. Strong anion-exchange chromatography (SAX) offers a robust approach for separating charged oligonucleotides without relying on volatile ion-pairing agents, making it attractive for large-scale and high-throughput applications.

Objectives and Study Overview

This study set out to optimize SAX separation parameters for DNA and RNA oligonucleotides ranging from 25 to 100 bases. Specific goals included evaluating the influence of mobile phase composition (buffer pH, salt concentration, organic modifier), column temperature, and gradient conditions. The workflow included SAX fractionation of crude samples followed by confirmation of purity using ion-pair reversed-phase LC/UV and LC/MS analysis.

Methodology and Used Instrumentation

Anion-exchange separation was performed on an Agilent PL-SAX 1,000 Å, 2.1×50 mm, 5 µm column. Mobile phases were prepared from Tris buffers (pH 8.0) with and without 10 % acetonitrile, or 10 mM NaOH (pH 12), combined with 2 M NaCl as eluent. SAX gradients were scaled for 10-minute runs at various temperatures (30 °C to 80 °C).

Used instrumentation:

• Agilent 1290 Infinity II LC system with high-speed pump, multisampler and diode array detector (bio-inert flow cell)
• Agilent 6530 Q-TOF LC/MS with Jet Stream ESI source
• Data acquisition and analysis via Agilent MassHunter Acquisition and BioConfirm software

Results and Discussion

Introducing 10 % acetonitrile into 10 mM Tris (pH 8.0) significantly improved peak shape and resolution compared to aqueous Tris buffer. Raising mobile phase pH to 12 with 10 mM NaOH further sharpened peaks, especially for longer oligos, but introduced depurination and truncation risks at elevated temperatures. Systematic temperature studies on a 105-mer sgRNA revealed that combining 10 % acetonitrile in Tris buffer with 80 °C column temperature yielded the narrowest peaks and suppressed secondary structure effects. SAX fractionation of crude 100-mer DNA followed by IP-RP LC/UV and LC/MS confirmed that early-eluting fractions contained n-5 truncated species, while the central fraction was enriched in full-length product. LC/MS deconvolution highlighted sodium adduct formation from the SAX eluent.

Benefits and Practical Applications

The optimized SAX method provides high separation efficiency, improved throughput, and reduced reliance on volatile ion-pair reagents. It enables reliable purification of oligonucleotides for therapeutic development, quality control in manufacturing, and research applications. The fractionation workflow coupled with orthogonal LC/UV and LC/MS confirmation ensures accurate identification of full-length and truncated sequences.

Future Trends and Potential Applications

Future developments may include integration of automated SAX fraction collectors, coupling to high-resolution MS for real-time impurity profiling, and expansion to modified or longer oligonucleotides. Advances in column chemistries and buffer systems could further enhance resolution and reduce degradation. The method holds promise for rapid screening of oligo libraries and scale-up in biopharmaceutical production.

Conclusion

This application note demonstrated a systematic approach to optimize SAX chromatography for oligonucleotides, highlighting the roles of organic modifier, pH, and temperature. The workflow combining SAX fractionation with IP-RP LC/UV and LC/MS verification delivers a robust strategy for obtaining high-purity synthetic oligos.

References

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3. Kanavarioti K. HPLC Methods for Purity Evaluation of Man-Made Single-Stranded RNAs. Sci Rep. 2019;9:1019.