HILIC as an Alternative Separation Mode for Intact Mass Confirmation of Oligonucleotides on the BioAccord System

Importance of the Topic

The rapid growth of oligonucleotide-based therapeutics demands robust methods for confirming intact molecular mass under native conditions. High performance workflows capable of delivering accurate mass data while reducing cost and toxicity are essential for manufacturing control and regulatory compliance.

Objectives and Study Overview

This study evaluates hydrophilic interaction chromatography (HILIC) coupled to a compact BioAccord LC-MS System as an alternative to ion-pair reversed-phase (IP-RP) for intact mass confirmation of oligonucleotides up to 57-mer. The aim is to assess mass accuracy, chromatographic performance, mobile phase stability, and overall workflow compliance.

Methodology

Oligonucleotide standards including polyT mixtures, a fully phosphorothioated 25-mer, and a 57-mer sequence were analyzed. Samples (10 μM) were separated using a HILIC gradient on an ACQUITY Premier BEH Amide column at 60 °C with ammonium acetate/acetonitrile mobile phases. Full-scan negative ESI MS data were acquired at 400–5000 m/z and processed using BayesSpray in waters_connect.

Instrumentation Used

• BioAccord LC-MS System with ACQUITY UPLC I-Class PLUS and TUV detector
• ESI-Tof ACQUITY RDa Mass Detector
• ACQUITY Premier BEH Amide 1.7 μm, 2.1×50 mm column with MaxPeak HPS technology

Main Results and Discussion

HILIC separation delivered stable UV responses and high recovery of polyT standards from the first injection, eliminating column passivation required in stainless steel hardware. ESI-MS spectra exhibited narrower charge distributions (3–4 states) indicative of native conformers and facilitated accurate deconvolution (mass errors <15 ppm). Phosphorothioated and 57-mer oligonucleotides achieved similar mass accuracy to IP-RP methods. Mobile phases showed consistent MS/UV signal ratios over two weeks, highlighting cost-effective and less toxic operation.

Benefits and Practical Applications

• Cost reduction by over 10× through elimination of expensive ion-pair reagents
• Reduced toxicity and improved laboratory safety
• Extended mobile phase stability (up to two weeks) enhancing throughput
• Native-like conformations preserve analyte integrity for downstream analyses

Future Trends and Opportunities

Emerging HILIC stationary phase designs and surface modifications will further mitigate metal interactions and broaden applications. Integration with automated compliance platforms and advanced deconvolution algorithms will enable high-throughput intact mass screening in quality control and biopharmaceutical research.

Conclusion

HILIC LC-MS on the BioAccord System provides a reliable, cost-effective alternative for intact mass confirmation of oligonucleotides. The approach delivers high mass accuracy, greater mobile phase stability, and reduced sample preparation, supporting robust analytical workflows for oligonucleotide therapeutics.

References

1. Sharma VK, Watts JK. Oligonucleotide Therapeutics: Chemistry, Delivery and Clinical Progress. Future Med Chem. 2015;7(16):2221-2242.
2. Roberts TK, Langer R, Wood MJA. Advances in Oligonucleotide Drug Delivery. Nat Rev Drug Discov. 2020;19:673-694.
3. Doneanu CE, Fox J, Harry E, et al. Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation of Oligonucleotides. Waters Application Note. 2020;720006820EN.
4. Doneanu CE, Fox J, Harry E, et al. Intact Mass Confirmation on the BioAccord LC-MS System for Modified Oligonucleotides. Waters Application Note. 2020;720007028EN.
5. Goyon A, Yehl P, Zhang K. Characterization of Therapeutic Oligonucleotides by LC. J Pharm Biomed Anal. 2020;182:1-17.
6. Sutton JM, Guimaraes GJ, Annavarapu V, et al. Oligonucleotide Characterization Using LC-MS. J Am Soc Mass Spectrom. 2020;31:1775-1882.
7. Lobue PA et al. Oligonucleotide Analysis by HILIC-MS without Ion-Pair Reagents. J Chromatogr A. 2019;1595:39-48.
8. Lauber M et al. Low Adsorption HPLC Columns based on MaxPeak HPS Surfaces. Waters White Paper. 2020;720006930EN.
9. DeLano M et al. Mitigation of Analyte Interactions with Metal Surfaces in UHPLC. Anal Chem. 2021;93:5773-5781.
10. Gilar M, DeLano M, Gritti F. Mitigation of Analyte Interactions with Metal Surfaces in UHPLC. J Chromatogr A. 2021;1650:462247.
11. Brennan K, Trudeau M, Rainville PD. ACQUITY Premier for Improved Oligonucleotide Performance. Waters App Note. 2021;720007119EN.
12. Koshel BM, Birdsall RE, Yu YQ. Improved Recovery of Oligonucleotide Impurities Using MaxPeak HPS. Waters App Note. 2021;720007238EN.
13. Alpert AJ. HILIC for Separation of Peptides, Nucleic Acids and Polar Compounds. J Chromatogr. 1990;499:176-196.
14. Hemstrom P, Irgum K. Hydrophilic Interaction Chromatography. J Sep Sci. 1990;499:176-196.
15. Gilar M et al. Kinetics of Phosphorothioate Oligonucleotide Metabolism. Nucleic Acids Res. 1997;25:3615-3620.
16. Zhang R, Diasio RB, et al. Pharmacokinetics of Oligonucleotide GEM 91. Biochem Pharmacol. 1995;49:929-939.
17. Birdsall R et al. Reduction of Metal Adducts in Oligonucleotide Mass Spectra. Rapid Commun Mass Spectrom. 2016;30:1667-1679.