Oligonucleotide Analysis Using the Agilent InfinityLab Pro iQ and Altura Oligo HPH-C18 Column

Oligonucleotide analysis using Altura Oligo HPH-C18 and Agilent InfinityLab Pro iQ — Application note summary

Significance of the topic

Oligonucleotide therapeutics (ASOs, siRNAs) are an expanding drug modality that demand stringent purity and identity control. Analytical workflows able to separate near-isobaric impurities (notably n-1 mers) and to confirm molecular weights at trace levels are essential for QC, process development, and regulatory filings. Combining chromatography that minimizes secondary interactions with MS-friendly conditions improves sensitivity and reduces method development complexity for biopharma laboratories.

Objectives and overview of the study

This application note evaluated a combined LC–MS workflow for oligonucleotide impurity profiling that prioritizes MS compatibility while preserving chromatographic resolution. The study compared the Agilent Altura Oligo HPH‑C18 column (ultra‑inert hardware) against the AdvanceBio Oligonucleotide column, paired with an Agilent 1260 Infinity III Prime bio LC and the InfinityLab Pro iQ mass detector. Key goals were to: maintain or improve separation of closely eluting species at reduced ion‑pair concentrations, obtain robust MS spectra across relevant charge envelopes, and enable deconvolution-based molecular weight confirmation of main components and impurities.

Methodology and instrumentation

The study employed two sets of mobile phase conditions: a high ion‑pair concentration method (100 mM hexylammonium acetate, HAA) optimized for UV separation and an MS‑friendly method (15 mM hexylamine, HA, with 25 mM HFIP) designed to minimize MS suppression. Chromatography conditions common to the experiments included 2.1 × 150 mm, 2.7 μm columns, 0.6 mL/min flow, 60 °C column temperature, 20 μL injections, and UV detection at 260 nm. Gradients were adjusted to the respective mobile phases to elute ladder and resolution standards.

Instrumentation

• Agilent 1260 Infinity III bio flexible pump (G7131C)
• Agilent 1290 Infinity III bio multisampler with thermostat (G7137C)
• Agilent 1290 Infinity III multicolumn thermostat with Quick Connect bio heat exchanger (G7116B)
• Agilent 1260 Infinity III diode array detector with Max‑Light 10 mm cell (G7117C)
• Agilent Altura Oligo HPH‑C18 column, 2.1 × 150 mm, 2.7 μm (227215‑702)
• Agilent AdvanceBio Oligonucleotide column, 2.1 × 150 mm, 2.7 μm (653750‑702)
• InfinityLab Pro iQ mass detector (G6160B), ESI negative mode; scan m/z 500–1,600; gas temp 325 °C; gas flow 11 L/min; nebulizer 45 psi; capillary 4,500 V; fragmentor: stepped/ramped voltages
• Data processing: Agilent OpenLab CDS v2.8 (feature pack 2)

Main results and discussion

- Under 100 mM HAA (UV‑optimized), both columns delivered comparable separation of 39 nt and 40 nt oligo dT species, showing that high ion‑pair concentration compensates for hardware differences.

- Under MS‑friendly conditions (15 mM HA + 25 mM HFIP), the Altura Oligo HPH‑C18 column maintained high resolution between the 39 and 40 nt species comparable to the 100 mM HAA method, whereas the AdvanceBio column showed a marked loss of resolution for this pair. This demonstrates that ultra‑inert hardware substantially reduces non‑specific interactions and peak broadening when ion‑pair concentration is lowered.

- The Altura column also produced higher UV peak intensities than the AdvanceBio column at equivalent loadings under low‑ion‑pair conditions, suggesting improved recovery and reduced adsorption losses.

- MS performance on the InfinityLab Pro iQ was adequate to capture multiply charged envelopes of oligonucleotides (major charge states concentrated between m/z ~500–1,300). The instrument’s scan range and speed provided clean raw spectra and enabled deconvolution into accurate molecular weights for ladder components from ~4.5 to >12 kDa.

- A critical demonstration was the resolution and MS characterization of the 39 nt n‑1 impurity adjacent to the 40 nt main peak. Deconvolution revealed a mass difference of 304 Da relative to the full‑length 40 nt sequence, allowing unambiguous identification of the truncation.

Benefits and practical applications of the method

• Ability to operate under MS‑compatible ion‑pair concentrations (15 mM HA) while preserving resolution reduces MS signal suppression and instrument contamination.
• Ultra‑inert column hardware minimizes secondary interactions, improving peak shape, recovery, and impurity detection—critical for n‑1 and near‑isobaric species.
• Lower reagent consumption and decreased chemical noise reduce operational costs and maintenance burden.
• The Pro iQ detector provides sufficient m/z coverage and scan speed to support deconvolution workflows for oligonucleotide identity confirmation across a broad mass range.
• Workflow supports impurity profiling, release testing, and method transfer for therapeutic oligonucleotides.

Future trends and potential applications

• Broader adoption of ultra‑inert column hardware for ion‑pair reversed‑phase methods to improve MS compatibility across oligonucleotide analytics.
• Continued development of volatile ion‑pair chemistries and alternatives (e.g., ion‑exchange, HILIC, enzymatic‑based assays) to reduce MS suppression while maintaining resolution.
• Integration with higher‑resolution and faster MS systems (orbitrap, TOF) and improved deconvolution algorithms to increase confidence in low‑abundance impurity assignment and sequence modification mapping.
• Automation and standardized LC–MS workflows for routine QC, batch release, and regulatory submissions.
• Application to longer and more heavily modified oligonucleotides as therapeutics evolve, including conjugates and chemically modified backbones.

Conclusion

The combination of the Altura Oligo HPH‑C18 column with ultra‑inert hardware and the Agilent InfinityLab Pro iQ mass detector provides a practical, MS‑compatible workflow that preserves chromatographic resolution at reduced ion‑pair concentrations. This enables sensitive MS detection, robust deconvolution, and confident molecular weight confirmation of main oligonucleotides and closely eluting impurities such as n‑1 mers. The approach reduces reagent use and instrument contamination while supporting rigorous impurity profiling needed in therapeutic oligonucleotide development and QC.

References

1. Roberts, T. C.; Langer, R.; Wood, M. J. A. Advances in Oligonucleotide Drug Delivery. Nature Reviews Drug Discovery 2020, 19(10), 673–694.
2. Sutton, J. M.; Guimaraes, G. J.; Annavarapu, V.; van Dongen, W. D.; Bartlett, M. G. Current State of Oligonucleotide Characterization Using Liquid Chromatography–Mass Spectrometry: Insight into Critical Issues. Journal of the American Society for Mass Spectrometry 2020, 31(9), 1775–1782.
3. Chae‑Young, R.; Brian, L. Oligonucleotide Analysis with Ion‑Pair Reversed‑Phase Chromatography and Agilent 1260 Infinity II Prime LC. Agilent Technologies Application Note 2022, Publication 5994‑5323EN.
4. Studzińska, S.; Rola, R.; Buszewski, B. The Impact of Ion‑Pairing Reagents on the Selectivity and Sensitivity in the Analysis of Modified Oligonucleotides in Serum Samples by Liquid Chromatography Coupled with Tandem Mass Spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2017, 138, 146–152.