Comprehensive characterization of oligonucleotides and related impurities using advanced LC/Q-TOF analytical workflows

Importance of the topic

Therapeutic oligonucleotides are an expanding class of modalities with sequence-specific biological activity but substantial structural heterogeneity. Robust impurity profiling is essential for safety, efficacy and regulatory compliance because common manufacturing- and storage-related variants (backbone modifications, truncations/extensions, depurination, abasic sites, and PS→PO conversions) can alter pharmacology and immunogenicity. Advanced analytical workflows that combine high-resolution chromatography and accurate-mass MS enable characterization of both major product and low-level related impurities that often coelute in conventional one-dimensional LC/MS methods.

Objectives and overview of the study

This study demonstrates a comprehensive 2D-LC/MS workflow to characterize nusinersen (an antisense oligonucleotide, ~7.127 kDa) and its related impurities. Goals were to (1) resolve coeluting isoforms and low-abundance variants, (2) provide MS and MS/MS-based sequence confirmation of full-length product (FLP) and truncated/modified species, and (3) improve sensitivity through MS-compatible desalting in a second dimension while maintaining throughput via multi-heart-cut/high-resolution sampling.

Methodology

The workflow uses orthogonal two-dimensional LC with targeted multiple-heart-cut (MHC) transfers into a desalting/analytical 2D column followed by accurate-mass Q-TOF detection and automated sequence confirmation.

• First-dimension separation: ion-pairing reversed-phase (Altura Oligo HPH-C18) using dibutylamine/HFIP-containing buffer to maximize chromatographic resolution of sequence and backbone isomers.
• Second-dimension desalting/separation: HILIC-Z (Altura Poroshell) with ammonium acetate-based mobile phase to provide MS-compatible conditions and enhance sensitivity for transferred heart-cuts.
• Sampling strategy: HiRes sampling combined with a multi-inject approach (biocompatible loops emulating large loop volumes) to transfer multiple heart-cuts per run, improving throughput while preserving high 1D separation fidelity.
• MS acquisition and data processing: Agilent 6545XT AdvanceBio Q-TOF operated in negative ion mode with wide mass range (m/z ~400–3,200) and MS/MS collection. MassHunter BioConfirm software was used for Target+Impurities and Sequence Confirmation workflows with tolerances set typically to ±10 ppm for MS and ±20 ppm for MS/MS.

Used instrumentation

• Agilent 1290 Infinity III Bio 2D-LC platform (flexible and high-speed pumps, multisampler, multicolumn thermostat, diode-array/UV detectors) configured for MHC 2D operation.
• Columns: Altura Oligo HPH-C18 (4.6 × 150 mm, 2.7 μm) for 1D; Altura Poroshell HILIC-Z (2.1 × 100 mm, 2.7 μm) for 2D.
• Mass spectrometer: Agilent 6545XT AdvanceBio LC/Q-TOF. Representative MS settings included source gas temperatures ~250–350 °C, drying and sheath gas flows ~12–13 L/min, VCap ~4,500 V, nozzle ~2,000 V, fragmentor ~180 V, and negative-mode acquisition.
• Software: MassHunter BioConfirm for automated sequence annotation, impurity detection and MS/MS-based confirmation.

Main results and discussion

• The 2D-LC approach effectively separated coeluting species that were unresolved in 1D runs. Heart-cut transfers of the 1D peak region containing the FLP and coeluting impurities enabled their chromatographic resolution on the HILIC-Z 2D column.
• Mass spectra and MS/MS fragmentation ladders (y, b and w ions) allowed unambiguous identification of the FLP and multiple truncated variants (e.g., N-1 truncations) and chemical variants such as phosphorothioate (PS) to phosphodiester/PO conversions. MS/MS fragmentation patterns localized modifications and termini-specific changes (5′/3′ signatures).
• HiRes multi-inject sampling transferred multiple targeted heart-cuts (e.g., 10 heart-cuts per run) improving throughput while enabling high-resolution MS characterization of trace-level peaks across the chromatogram.
• Mass accuracy and MS/MS data quality from the 6545XT provided confident identifications of low-abundance impurities; automated BioConfirm workflows streamlined matching observed masses/fragment ions to expected sequences with defined mass tolerances.

Benefits and practical applications of the method

• Enhanced impurity detection: Orthogonal 2D separations plus MS-compatible desalting improve signal-to-noise and permit detection/confirmation of low-level sequence variants and chemical degradants.
• Regulatory relevance: The approach supports impurity profiling required for GMP and therapeutic development by delivering sequence-level evidence for product-related and process-related variants.
• Throughput and flexibility: Multi-heart-cut and multi-inject strategies preserve 1D resolution while increasing sample throughput and enabling targeted analysis of regions of interest without hardware modification.
• Automated data interpretation: BioConfirm workflows reduce manual annotation time and provide reproducible criteria for sequence confirmation and impurity assignment.

Future trends and possibilities for application

• Increased automation and standardized 2D-LC/MS methods for routine GMP release and stability testing of oligonucleotide therapeutics.
• Integration with ion mobility spectrometry to add an additional orthogonal separation dimension for isomeric/isobaric species.
• Higher-resolution and faster MS platforms to push limits of detection for ultra-trace impurities and enable more extensive MS/MS mapping of modifications.
• Improved software workflows incorporating machine learning for deconvolution of complex charge envelopes and automated localization of chemical modifications.
• Expansion toward quantitative 2D-LC/MS methods and validated impurity assays for regulated environments, and adoption for novel modalities such as siRNA, gapmers and conjugated oligonucleotides.

Conclusion

The presented 2D-LC/MS workflow combining ion-pair RP 1D separations with HILIC-Z MS-compatible 2D desalting, multi-heart-cut sampling and accurate-mass Q-TOF detection provides a practical, high-resolution platform for comprehensive characterization of oligonucleotide therapeutics. The approach resolves coeluting isoforms, identifies truncations and backbone modifications by MS/MS, and increases sensitivity for low-abundance impurities while maintaining throughput through multi-inject strategies. Coupling robust chromatographic design with automated sequence-confirmation software delivers a scalable solution for development and quality control of oligonucleotide drugs.

Reference

Agilent Technologies. Analysis of GLP-1 Agonists Using the Agilent 1290 Infinity II Bio LC System and Altura Ultra Inert HPLC Column. Agilent publication number 5994-8837EN, 2025.