Scalable Purification of a Synthetic Oligonucleotide Using Agilent PL-SAX Columns

Importance of the Topic

Synthetic oligonucleotides are crucial tools in modern biopharmaceutical research, with applications ranging from gene regulation to therapeutic interventions. Achieving high purity and yield of these negatively charged molecules is essential for downstream biological efficacy and regulatory compliance. Anion-exchange chromatography (AEX) using strong anion-exchange (SAX) media provides a robust, scalable, and environmentally friendly alternative to ion-pair reversed-phase methods, avoiding toxic volatile additives and residual counter-ions.

Study Objectives and Overview

This application note describes the development and scale-up of an AEX purification protocol for a crude, all-2′-O-methylated 22-mer RNA oligonucleotide. The study aims to demonstrate consistent separation performance on both analytical and preparative scales using Agilent PL-SAX columns, evaluate binding capacity, and define fraction collection strategies to maximize combined yield and purity.

Used Instrumentation and Methodology

• Analytical system: Agilent 1290 Infinity III LC with PL-SAX 1000Å 4.6×150 mm, 8 µm column; diode array detection at 260 nm; flow rate 1.0 mL/min; gradient 20→80% B over 30 min at 6 °C.
• Preparative system: Agilent 1290 Infinity II preparative LC with PL-SAX 1000Å 25×150 mm, 10 µm column; gradient 20→100% B over 27 min at 25 °C; flow rate 29.5 mL/min; fraction collection in 1.5 mL intervals.
• Buffers: Eluent A – 10 mM Tris-HCl pH 8.0; Eluent B – 10 mM Tris plus 1 M NaCl. Reanalysis by IP-RP LC using TEAA/ACN on AdvanceBio oligonucleotide column.
• Software: Agilent OpenLab 2.8.

Main Results and Discussion

Initial analytical separation of the crude sample yielded approximately 85% purity under the scouting gradient. Scale-up to preparative dimensions maintained resolution, with linear scaling of flow rate and injection volume demonstrating dynamic binding capacity of ~12 mg/mL. A 300 µL injection (6 mg) and an 800 µL injection (16 mg) produced well-resolved main peaks without column overload. Sixteen fractions were collected and reanalyzed by IP-RP, revealing peak purities up to 97% across fractions F03–F11. Combining optimal fractions led to an overall recovery of ~96% purity and high cumulative yield.

Benefits and Practical Applications

AEX on PL-SAX columns eliminates the need for hazardous fluorinated additives and organic solvents, reducing both environmental impact and downstream cleanup steps. The method is directly transferable from analytical QC to preparative manufacturing, offering high binding capacity and buffer compatibility. This approach is suitable for routine purification of therapeutic oligonucleotides at laboratory and production scales.

Future Trends and Opportunities

Continued development of AEX media with enhanced selectivity and binding capacity promises further improvements in throughput and resolution. Integration of bulk PL-SAX resins into continuous-flow systems could support large-scale API production. Advances in automated fraction analysis and inline monitoring will accelerate process optimization for diverse oligonucleotide chemistries, including heavily modified or longer sequences.

Conclusion

The study validates a scalable, robust AEX protocol for the purification of synthetic oligonucleotides using Agilent PL-SAX columns. Analytical and preparative separations yield high purity and recovery, demonstrating straightforward method transfer and operational efficiency without relying on volatile or toxic additives.

References

1. Hsiao J, Apffel A, Turner M. Optimizing Separation of Oligonucleotides with Anion-Exchange Chromatography. Agilent Technologies Application Note 5994-4753EN, 2022.
2. Massi J, Lloyd L. High Resolution Separations of Oligonucleotides using PL-SAX Strong Anion-Exchange HPLC Columns. Agilent Technologies Application Note 5990-8297EN, 2021.
3. Tripodi AAP, Coffey A. Superficially Porous Columns for Semi-Preparative Purification of Oligonucleotides. Agilent Technologies Application Note 5994-7478EN, 2024.