Oligonucleotide sequencing by LC-MS/MS

Significance of the Topic

Messenger RNA (mRNA) vaccines and oligonucleotide therapeutics have revolutionized modern medicine by providing rapid and adaptable platforms for disease prevention and gene modulation. Ensuring their sequence integrity and chemical identity is critical for safety, efficacy and regulatory compliance. Advanced analytical workflows that combine high‐resolution separation with precise mass spectrometric sequencing enable thorough characterization and quality control of these complex biomolecules.

Objectives and Study Overview

This study presents a comprehensive LC‐MS/MS approach for sequencing oligonucleotides ranging from short RNAs to long mRNA strands. Key goals include:

• Developing a robust partial digestion protocol to generate sequenceable fragments of optimal length.
• Optimizing ion‐pair chromatography conditions to achieve high resolution across 10–60 nucleotide fragments.
• Refining mass spectrometry settings and collision energy regimes to maximize sequence coverage and minimize adduct formation.
• Demonstrating reproducibility and practical applicability as a quality control method for mRNA vaccines.

Methodology and Instrumentation

Sample Preparation and Digestion:

• Immobilized RNase T1 magnetic beads enable controlled, partial digestion of single‐stranded regions, delivering reproducible fragment patterns and structural insights into RNA folding.
• Bead removal halts digestion rapidly, preventing over‐cleavage and column contamination.

Chromatography and Ion Pairing:

• DNAPac RP UHPLC columns operated on a Vanquish UHPLC system provide high‐efficiency separation.
• Triethylamine (TEA) with varying concentrations of 1,1,1,3,3,3‐hexafluoroisopropanol (HFIP) forms ion pairs with the phosphate backbone, balancing retention and peak shape. Higher HFIP levels extend retention and narrow charge state distributions.

Mass Spectrometry:

• Orbitrap Exploris mass spectrometer in negative ion mode acquires full scans at 120,000–240,000 resolution depending on fragment length.
• Stepped normalized collision energies (NCE) tailored to fragment size (e.g., 10–12–14 for shorter oligos, 18–20–22 for impurity analysis) enhance fragmentation efficiency and sequence coverage.

Data Analysis:

• BioPharma Finder software automates ribonuclease selection, chromatogram processing, sequence mapping and percent coverage calculations, supporting rapid confirmation of expected mRNA sequences.

Main Results and Discussion

Fragmentation Optimization:

• Optimal HFIP/TEA ratios yield sharp separation of 10–60 mers, crucial for resolving isomeric sequences sharing identical monoisotopic masses.
• Lower stepped NCE regimes produced higher Average Structural Resolution (ASR) values for small oligos, whereas moderate energies improved long‐oligo fragmentation without excessive internal cleavage.

Partial Digest Performance:

• Immobilized RNase T1 partial digests of spike and eGFP mRNA demonstrated highly reproducible fragment patterns across replicates, enabling ultraviolet or MS‐based QC assays.
• Fragment distribution reflected mRNA secondary structure, providing additional conformational information.

Sequence Isomer Separation:

• Chromatographic method separated sequence isomers with identical 5131.6658 Da mass, highlighting the system’s specificity.

Benefits and Practical Applications

This LC‐MS/MS workflow offers:

• Routine sequencing of oligonucleotides up to 60 nt with >99% confidence in fragment assignment.
• Detection of known chemical modifications and low‐level impurities.
• A streamlined 60-minute protocol from sample preparation to data analysis, suitable for QA/QC laboratories.
• Scalability to diverse mRNA vaccine platforms and other oligonucleotide therapeutics.

Future Trends and Applications

Emerging directions include:

• Integration of alternative nucleases or engineered enzymes for complementary fragmentation patterns.
• Enhanced software algorithms and machine learning to improve de novo oligonucleotide sequencing and identify novel modifications.
• Automation of sample preparation and data processing in high-throughput formats.
• Extension of the platform to long non-coding RNAs and combination therapeutics containing multiple oligonucleotide species.

Conclusion

The described LC-MS/MS strategy combining immobilized RNase T1 partial digestion, optimized ion-pair UHPLC and stepped HCD fragmentation on an Orbitrap mass spectrometer provides a powerful, reproducible approach for comprehensive oligonucleotide sequencing. Its implementation supports stringent quality control of mRNA vaccines and biotherapeutics, delivering high sequence coverage, structural insight and rapid turnaround.

Reference

Vanhinsbergh C. et al. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. Anal. Chem. 2022, 94(20), 7339–7349.