Cation exchange coupled to high resolution mass spectrometry for the analytical characterization of phosphorodiamidate morpholino oligomers

Cation exchange coupled to high-resolution MS for analytical characterization of phosphorodiamidate morpholino oligomers (PMOs) — concise expert summary

Significance of the topic:

Phosphorodiamidate morpholino oligomers (PMOs) are an emerging class of antisense therapeutics with uncharged phosphorodiamidate linkages and morpholine rings that render conventional oligonucleotide analytics inadequate. Reliable, sensitive and specific impurity profiling of PMOs is critical for quality control, regulatory submission and process development because manufacturing generates unique degradation and side-product profiles that affect safety and efficacy. Developing robust workflows able to separate and identify low‑level impurities, including deamination products, is therefore a practical priority for biopharmaceutical analytics.

Objectives and study overview:

This work presents a novel direct-analysis platform that couples cation-exchange liquid chromatography (CEX) using a volatile pH gradient to positive‑ion high-resolution mass spectrometry (HRMS). The method aims to (1) exploit the amine-containing backbone of PMOs to achieve sequence- and size-selective separation by isoelectric focusing along a pH gradient, (2) enable direct HRMS identification and quantitation of full-length product (FLP) and a comprehensive set of impurities (amidate, backbone fragments, N-1/N+1 species, base losses, oxidation, alkylation, deamination, tail-off products), and (3) deliver a platform workflow compatible with routine GLP laboratory practice.

Methodology (conceptual):

The chromatography uses a ProPac CEX stationary phase and a volatile eluent system to create a rising pH gradient. At low pH PMOs are positively charged and bind the column; as pH increases each species reaches its isoelectric point and elutes. Eluates are detected by positive-ion HRMS (Orbitrap) operated with optimized source conditions to minimize adducts while avoiding in‑source fragmentation. Data processing applies sliding-window deconvolution to obtain intact masses for each chromatographic peak; identified m/z species are transferred to a component/XIC table in Chromeleon for targeted quantitation and reporting. The workflow supports both untargeted (deconvolution-first) and targeted (XIC) analyses for compliant impurity measurement.

Used instrumentation:

• UHPLC: Thermo Scientific Vanquish Flex Binary UHPLC with Thermo Scientific ProPac 3R cation-exchange column (3 µm, 2.1 × 50 mm).
• Mass spectrometer: Thermo Scientific Orbitrap Exploris high-resolution MS (positive-ion detection; resolution up to 120,000 used for isotope clarity).
• Software/data handling: Thermo Scientific Chromeleon 7.3.2 / 7.4 with CM reporting engine 2.0, sliding-window deconvolution and component/XIC transfer.

Key method development points:

• Volatile pH-gradient elution was chosen to modulate PMO charge and enable direct coupling to MS without ion-pair reagents.
• Source in‑source collision energy was optimized to 30 eV to reduce adduct formation but avoid artifactual base-loss fragments; higher energies (50–70 eV) produced in‑source degradation and were excluded.
• Deconvolution parameters and targeted XICs were tuned to provide both sensitive detection and compliant quantitative reporting.

Main results and discussion:

• The same chromatography and MS settings successfully profiled four pharmaceutically relevant PMOs (golodirsen, casimersen, viltolarsen, eteplirsen), demonstrating platform applicability across different sequences.
• Chromatography showed both sequence-selective and size-selective separation: many impurity types exhibited characteristic retention times reproducible between samples, enabling retention-time assisted identification.
• Intact-mass deconvolution combined with retention-time confirmation permitted assignment of a broad set of impurities: N-1 and N+1 base variants, amidate (-27 Da), deamination, -44 Da species, CNET, +106 Da, oxidation products, guanine base loss, +123 Da species, tail-off products and others.
• Deamination — a key chemical degradation pathway — was chromatographically separated from the FLP and quantified by MS; authors report this as the first direct measurement of PMO deamination using ion-exchange coupled to HRMS.
• Annotated high-resolution spectra (120,000) delivered clear isotope envelopes and low-background spectra because each impurity entered the MS separated from other species.
• Sensitivity and precision: limits of detection were reported below ~0.02% fractional abundance. Replicate injections (example: casimersen) showed excellent reproducibility; even low-level deamination (~0.46% fractional abundance) had an RSD of 2.8%, indicating high precision for impurity profiling.
• The volatile eluent system is described as more environmentally friendly (no persistent ion-pairing reagents) and the workflow is compatible with GLP reporting using Chromeleon.

Practical benefits and applications:

• Enables direct, high-sensitivity impurity profiling of PMOs at intact-mass level without prior derivatization or non-volatile mobile phases.
• Provides sequence- and size-sensitive separation, improving confidence in impurity identification when combined with HRMS deconvolution.
• Combines untargeted discovery (deconvolution) with targeted quantitation (XIC) in a single run for regulatory-friendly workflows.
• Applicable across multiple PMO candidates using a single set of LC–MS parameters, facilitating platformization for QC and process development labs.

Limitations and considerations:

• Method performance depends on careful source optimization to avoid in‑source fragmentation artifacts that could be misassigned as real impurities.
• While HRMS affords clear isotope profiles and high sensitivity, implementation on lower-resolution instruments would require validation of specificity and limits of detection.
• Assignment of some modifications relies on delta-mass and retention behavior; confirmatory fragmentation (MS/MS) could be required for structural proof in certain regulatory contexts.

Future trends and applications:

• Extension of the method to other neutral or modified oligonucleotide modalities (e.g., other backbone-modified antisense chemistries) where ion-exchange exploiting amine functionalities can be used.
• Integration with two-dimensional LC (e.g., CEX × reversed-phase or ion pairing) for improved separation of complex impurity profiles where necessary.
• Application of automated deconvolution and AI-assisted annotation to accelerate impurity identification and reduce manual review.
• Adaptation for routine QC on lower-resolution MS platforms after method bridging and validation, enabling broader deployment in manufacturing environments.
• Use in forced-degradation and stability studies, in-process control, and comparability testing to support regulatory submissions.

Conclusions:

This study describes a practical, sensitive and reproducible CEX–HRMS workflow tailored for PMO intact-mass impurity profiling. By exploiting the amine-containing backbone and a volatile pH-gradient elution, the method achieves sequence- and size-selective separation, clean positive-ion HRMS detection, and confident deconvolution-based identification of a comprehensive range of PMO impurities, including the first reported direct quantitation of deamination in PMOs. The platform approach, combined with Chromeleon reporting, supports GLP-compliant workflows and promises broad utility across PMO development, QC and stability testing.

References:

1. Wei T.; Wang M.; Cai B.Z. Development of a two-dimensional liquid chromatography/mass spectrometry workflow for characterization of phosphorodiamidate morpholino oligomers. Journal of Chromatography A. 2025; 1757:466129.

Acknowledgements:

The authors of the original work acknowledged Thermo Fisher Scientific EU applications support for project execution and used Thermo instrumentation and software throughout their workflow.