Oligonucleotide Mass Confirmation and Impurity Identification

Importance of the Topic

Oligonucleotides serve as pivotal components in molecular diagnostics and therapeutics due to their modifiable chemistry and sequence specificity. Reliable confirmation of molecular weight and identification of impurities ensures product quality, safety, and efficacy in research, development, and manufacturing contexts.

Objectives and Study Overview

This work demonstrates the application of the Agilent InfinityLab LC/MSD XT system coupled with OpenLab CDS 2.8 spectral deconvolution for accurate molecular weight confirmation and impurity profiling across diverse synthetic oligonucleotide samples, including RNA standards, antisense oligonucleotides, aptamers, and cloning primers.

Methodology and Instrumentation

A reverse-phase ion-pairing LC method employing 15 mM triethylamine and 100 mM HFIP with a methanol gradient on an AdvanceBio Oligonucleotide column provided chromatographic separation. Detection was performed by diode array at 260 nm. Mass spectrometric analysis in negative ion mode used the Agilent InfinityLab LC/MSD XT, with deconvolution parameters optimized for charge state distribution and mass range.

Instrumentation

• Agilent 1290 Infinity II bio LC system with high-speed pump, multisampler with cooler, multicolumn thermostat, and diode array detector
• Agilent InfinityLab LC/MSD XT single quadrupole MS detector
• Agilent OpenLab CDS version 2.8 with MS spectral deconvolution

Main Results and Discussion

Separation of RNA standards yielded baseline resolution for 14-, 17-, 20-, and 21-mer oligos with mass accuracy within ±0.5 Da. Five diverse oligonucleotide samples ranging from 4 to 12 kDa showed consistent retention order related to length and chemical modifications, with mass deviations below 0.3 Da. A heterogeneous 38-mer cloning primer containing three coeluting sequence variants was deconvoluted to confirm individual variant masses. Impurity profiling of an ASO-21mer revealed n-1 to n-9 truncation series and various adducts, quantified by UV area percent with RSDs <5% and mass accuracy within ±0.5 Da.

Benefits and Practical Applications

The combined LC/MSD XT and automated deconvolution workflow offers cost-effective, robust, and high-throughput molecular weight confirmation and impurity identification for oligonucleotide QC. The approach enhances data processing efficiency and supports regulatory compliance in biopharma pipelines.

Future Trends and Applications

Advancements in deconvolution algorithms, integration with high-resolution MS platforms, and expanded workflows for deeper impurity profiling are anticipated. Enhanced automation and data analytics will further streamline QC processes for next-generation oligonucleotide therapeutics.

Conclusion

The Agilent InfinityLab LC/MSD XT system combined with OpenLab CDS deconvolution reliably confirms oligonucleotide molecular weight and identifies low-level impurities with high mass accuracy and reproducibility, providing a versatile platform for quality control in oligonucleotide analysis.

References

1. Agilent Technologies Application Note Oligonucleotide Characterization by Agilent 1290 Infinity II Bio LC and 6545XT AdvanceBio LC Q-TOF, publication number 5994-5788EN, 2023.
2. Agilent Technologies Application Note Unit Mass Spectral Deconvolution for Molecular Weight Confirmation of Large Molecules, publication number 5994-6928EN, 2024.
3. Capaldi D, Teasdale A, Henry S, Akhtar N, den Besten C, Gao-Sheridan S, et al. Impurities in Oligonucleotide Drug Substances and Drug Products. Nucleic Acid Ther 2017 27(6) 309–322.
4. Pourshahian S. Therapeutic Oligonucleotides, Impurities, Degradants and Their Characterization by Mass Spectrometry. Mass Spectrom Rev 2021 40(2) 75–109.