Identification and quantitation of oligonucleotides, impurities, and degradation products

Importance of the Topic

Oligonucleotide therapeutics are emerging modalities for treating genetic and infectious diseases. Accurate identification and quantitation of full-length sequences, synthesis-related impurities (shortmers, longmers, base-modified species), and degradation products are critical for drug discovery, quality control, and stability studies.

Objectives and Study Overview

The study aimed to develop a robust data-dependent MS2 (ddMS2) workflow for simultaneous identification, sequence mapping, and relative quantitation of oligonucleotide impurities in a single experiment. Three case studies demonstrate applications:

• Comparing impurity profiles of a DNA 21-mer purified by desalting versus HPLC.
• Characterizing degradation pathways under accelerated thermal and oxidative stress conditions.
• Elucidating structures of high-molecular-weight branched impurities.

Instrumentation

• Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer
• Thermo Scientific™ Vanquish™ Horizon UHPLC system
• Thermo Scientific™ DNAPac™ RP column (2.1 × 50 mm, 4 µm)
• Thermo Scientific™ BioPharma Finder™ 4.0 software

Methodology

Ion-pair reversed-phase UHPLC was performed at 60 °C with HFIP/DIPEA modifiers and a 15 min gradient (10–80 % methanol). The mass spectrometer operated in negative-ion mode with high-resolution full scans (120,000 at m/z 200) and data-dependent MS2 on charge states of interest. A stepped normalized collision energy (NCE) approach was optimized (18–20–22 % for 21-mer) to maximize fragment coverage. BioPharma Finder’s Oligonucleotide Analysis workflow was used for sequence mapping, deconvolution of intact masses, and comparative quantitation across multiple runs.

Main Results and Discussion

• Optimization of stepped NCE across eleven settings yielded complete fragment coverage of the 21-mer and its shorter impurities (average structural resolution ≤ 1.2).
• In desalting-purified samples, numerous shortmers (n–1 to n–19) were detected down to 0.015 % abundance; HPLC purification reduced total impurity from 2.55 % to 1.18 %.
• Forced thermal degradation at 80 °C led to rapid loss of full-length oligo within 4–6 h, accumulation of shortmers, phosphorylated species, base-loss, depurinated/depryrimidinated fragments, and nearly complete degradation by 24 h.
• Oxidative stress (5 % H2O2) produced stable full-length 21-mer levels but increasing oxidized species over 24 h, with minor changes in shortmer profiles.
• High-molecular-weight impurities (M + n–x) were observed around 9 min retention time only in desalting samples. MS1 deconvolution matched theoretical masses within < 1 ppm. ddMS2 fragmentation and mapping indicated a branched architecture linking shortmers via their 5′ ends to the 3′ base of the full-length oligo.

Benefits and Practical Applications

• Combines intact-mass confirmation, sequence-level mapping, and relative quantitation in one LC-MS run.
• Detects trace-level impurities below chromatographic visibility.
• Enables rapid assessment of purification methods and stability profiles for therapeutic oligonucleotides.
• Provides structural insights into complex branched impurities and degradation products.

Future Trends and Opportunities for Use

• Extension of ddMS2 workflows to longer or chemically modified oligonucleotides and siRNA constructs.
• Integration with automated reporting and high-throughput impurity screening pipelines.
• Advances in software algorithms for improved fragment assignment and quantitation accuracy.
• Application to in-process monitoring and release testing in oligonucleotide manufacturing.

Conclusion

The combined high-resolution accurate-mass/ddMS2 approach and BioPharma Finder software deliver a streamlined, sensitive, and comprehensive platform for characterizing oligonucleotides, their synthesis impurities, and degradation products. This methodology accelerates quality control, purity assessment, and structural elucidation in therapeutic oligonucleotide development.

Reference

1. Wang F et al. RNA therapeutics on the rise. Nat Rev Drug Discov. 2020;19:441.
2. Bajan S, Hutvagner G. RNA-based therapeutics: from antisense oligonucleotide to miRNAs. Cells. 2020;9:137.
3. Rossi JJ et al. Oligonucleotides and the COVID-19 pandemic: a perspective. Nucleic Acid Ther. 2020;30:129.
4. Sutton JM et al. Current state of oligonucleotide characterization using LC-MS. J Am Soc Mass Spectrom. 2020;31:1775.
5. El Zahar NM et al. Chromatographic approaches for therapeutic oligonucleotide impurities. Biomed Chromatogr. 2018;32:e4088.
6. Pourshahian S. Therapeutic Oligonucleotides, impurities, degradants, and their characterization by MS. Mass Spectrom Rev. 2019;doi:10.1002/mas.21615.
7. Capaldi D et al. Impurities in oligonucleotide drug substances and products. Nucleic Acid Ther. 2017;27:309.
8. Liu HC et al. Oligonucleotide mapping using BioPharma Finder software. Thermo Fisher App Note 73789;2020.
9. Kurata C et al. High molecular weight impurities in synthetic phosphorothioate oligonucleotides. Bioorg Med Chem Lett. 2006;16:607.