Direct Analysis of In-Process Oligonucleotides Without Manual Purification

Significance of the Topic

The reliable and rapid analysis of synthetic oligonucleotides is critical for applications in molecular diagnostics, therapeutics and quality control. Conventional ion-pair reversed-phase chromatography requires manual desalting when analyzing high-salt purification fractions, adding time and variability to workflows. Integrating online desalting with two-dimensional liquid chromatography streamlines analysis and improves reproducibility.

Goals and Overview of the Study

This study demonstrates a heart-cutting 2D-LC method combining online desalting in the first dimension with ion-pair reversed-phase separation in the second dimension. The objectives were to compare direct 2D-LC analysis of high-salt oligonucleotide solutions with traditional one-dimensional LC following manual desalting, evaluating speed, reproducibility and purity assessment.

Methodology

Oligonucleotide standards, an RNA/2’-O-methyl mix and a fully thiolated DNA sample were prepared in water and in 1 M NaCl to mimic anion-exchange purification fractions. Manual desalting employed centrifugal filters. The 2D-LC method used a PLRP-S column for online desalting in dimension one, heart-cut into an IP-RPLC column for separation in dimension two with active solvent modulation.

Used Instrumentation

• Agilent 1290 Infinity II 2D-LC System including two high-speed pumps, multisampler with cooler, dual multicolumn thermostats, dual diode array detectors and valve drives
• Agilent PLRP-S 100 Å, 2.1 × 50 mm, 3 µm column for online desalting
• Agilent AdvanceBio Oligonucleotide 2.1 × 50 mm, 2.7 µm column for IP-RPLC
• OpenLab CDS ChemStation with 2D-LC software

Main Results and Discussion

Direct 2D-LC achieved effective retention of oligonucleotides and removal of salts online, followed by clear IP-RPLC profiles with no peak splitting or breakthrough. Workflow time was reduced from approximately 96 minutes (manual desalting plus 1D separation) to 26 minutes (2D-LC), a more than threefold gain. Purity values of ~96 % matched those from manual methods. Retention time and peak area precision remained high in both dimensions.

Benefits and Practical Applications

• Eliminates labor-intensive manual desalting and associated sample loss
• Reduces total analysis time by over 70 %
• Enhances reproducibility through automated online processing
• Applicable to RNA, DNA and mixed oligonucleotide therapeutics

Future Trends and Opportunities

Further miniaturization and automation of 2D-LC workflows could enable high-throughput screening of oligonucleotide libraries. Coupling with mass spectrometry detection may broaden identification of impurities. The approach can be extended to other charged biomolecules and complex sample matrices.

Conclusion

Heart-cutting 2D-LC with online desalting provides a rapid, robust and automated solution for analyzing high-salt oligonucleotide samples. It significantly reduces workflow time, improves reproducibility and delivers purity results equivalent to manual methods.

References

1. Mangano M et al Composition dependent separation of oligonucleotides by capillary electrophoresis in acidic buffers with application to quality control of synthetic oligonucleotides Journal of Chromatography A 1999 848 435-442
2. Zimmermann A et al Synthetic oligonucleotide separations by mixed-mode reversed-phase/weak anion-exchange liquid chromatography Journal of Chromatography A 2014 1354 43-55
3. Mustonen P et al Oligonucleotide-based pharmaceuticals Non-clinical and clinical safety signals and non-clinical testing strategies Regulatory Toxicology and Pharmacology 2017 90 328-341
4. Shanagar Purification of a synthetic oligonucleotide by anion exchange chromatography Method optimization and scale-up Journal of Biochemical and Biophysical Methods 2005 64 216-225
5. Cramer F Herzberg Purity Analysis and Impurities Determination by Reversed-Phase High-Performance Liquid Chromatography In Handbook of Analysis of Oligonucleotides and Related Products CRC Press Taylor and Francis Group 2011 28-34