Rapid and Cost-Effective Desalting of HPLC-Purified Oligonucleotides

Significance of the Topic

Oligonucleotide purification is essential in biopharma and pharmaceutical research to achieve high analyte purity and reliable downstream analyses. Impurities such as salts or ion-pair reagents can interfere with mass spectrometry, cause signal suppression, and necessitate extended instrument cleaning. Developing rapid, high-yield desalting methods enhances laboratory throughput and data quality.

Objectives and Study Overview

This study evaluates the performance of centrifugal gel filtration using Agilent AdvanceBio Spin columns for desalting HPLC-purified oligonucleotides. A comparison is made against traditional ethanol precipitation in terms of speed, removal efficiency of ion-pair reagents (TEAA), and overall oligonucleotide recovery.

Methodology

The workflow consisted of four main steps:

• Preparative purification of a 22-mer 2′-O-methylated oligonucleotide by ion-pair reversed-phase HPLC (0.1 M TEAA mobile phase) and fraction collection.
• Pooled fraction analysis by analytical IP-RP HPLC to identify high-purity fractions.
• Desalting via two approaches: ethanol precipitation (multiple drying and wash steps) and centrifugal gel filtration with AdvanceBio Spin columns.
• Quantitative analysis of residual TEAA and oligonucleotide yield by size-exclusion chromatography (220 nm detection) and LC/MS using an ion-pair-free reversed-phase method.

Instrumentation

Preparative HPLC system:

• Agilent 1290 Infinity II preparative binary pump, fraction collector, column compartment, diode array detector.

Analytical IP-RP LC system:

• Agilent 1290 Infinity III high-speed pump, multisampler, multicolumn thermostat, diode array detector with Max-Light cartridge cell.

SEC analysis system:

• Agilent 1260 Infinity III bio-inert pump, multisampler, multicolumn thermostat, variable wavelength detector.

LC/MS system:

• Agilent 1290 Infinity II pump, multisampler, multicolumn thermostat, diode array detector; 6530 Q-TOF mass spectrometer.

Main Results and Discussion

Centrifugal gel filtration removed approximately 90 % of TEAA in under 10 minutes, cutting sample preparation time by 96 % compared to ethanol precipitation. The spin column approach yielded 84.5 % oligonucleotide recovery versus 46.5 % for ethanol precipitation. LC/MS analysis of spin-column-cleaned samples showed complete elimination of triethylammonium adduct signals and clear spectra of the full-length product with minor sodium and potassium adducts.

Benefits and Practical Applications

• Substantial reduction in sample preparation time and hands-on steps.
• Improved recovery and high purity of oligonucleotides.
• Enhanced compatibility with LC/MS, minimizing instrument downtime and maintenance.
• Scalable to semi-preparative workflows and adaptable to high-throughput laboratories.

Future Trends and Opportunities

Emerging ion-pair-free LC methods may further simplify oligonucleotide analysis. Advancements in spin-column materials could boost capacity and removal efficiency. Integration with automated platforms and coupling desalting directly with analytical instruments may streamline workflows in research and quality-control environments.

Conclusion

Agilent AdvanceBio Spin columns offer a rapid, high-yield, and effective desalting solution for HPLC-purified oligonucleotides. Compared to ethanol precipitation, they dramatically cut preparation time, maintain sample integrity, and ensure clean MS analysis, making them an attractive choice for modern analytical laboratories.

References

• Tripodi A.; Coffey A. Superficially Porous Columns for Semi-Preparative Purification of Synthetic Oligonucleotides. Agilent Technologies Application Note 5994-7478EN, 2024.
• Bertram L.; Hsaio J. Analysis of Oligonucleotides Using an Ion Pairing-Free Reversed Phase Method with TOF LC/MS. Agilent Technologies Application Note 5994-8013EN, 2024.