A novel monodisperse supermacroporous reversed-phase platform enabling unified LC–MS analysis of nucleic acids across a broad size range

Significance of the topic

Accurate, high-throughput LC–MS characterization of nucleic acid therapeutics is a growing analytical need across research, development and quality control. Nucleic acids cover a large size range (short antisense oligonucleotides to structured RNAs and mRNA), and common chromatographic media often perform well for either small or large species but not both. A unified reversed-phase LC–MS platform that provides reproducible separations and intact-mass information across a broad molecular-weight span simplifies workflows, reduces method proliferation, and supports robust method transfer and regulatory-compliant analyses.

Objectives and study overview

The study evaluated a novel 2.5 µm monodisperse supermacroporous (SMP) polymeric reversed-phase stationary phase (SurePac Oligo RP MDi) as a single, MS-compatible separation platform for nucleic acids spanning ~20 nucleotides (an antisense oligonucleotide, Tofersen) to ~75 nucleotides (yeast tRNAPhe). Goals were to demonstrate chromatographic performance, MS compatibility for intact-mass determination, and the platform’s ability to resolve sequence/chemical variants and adducts using rapid methods.

Methodology and used instrumentation

Key experimental elements and conditions were:

• Samples: Tofersen ASO (20 nt, 1 µg/µL injection at 1 µL) and baker’s-yeast tRNAPhe (~75 nt, 400 µg/µL injection at 1 µL).
• Column: Thermo Scientific SurePac Oligo RP MDi, 2.5 µm particle size, format 0.3 × 50 mm (P/N 43712050332), packed in hydrophobic-coated stainless steel inert hardware.
• Mobile phases: Solvent A = 15 mM dibutylamine (DBA) and 20 mM HFIP, pH 9.0; Solvent B = acetonitrile. DBA/HFIP ion-pairing enabled MS-compatible reversed-phase separations.
• Flow and temperature: 25 µL/min, column temperature 50 °C.
• LC methods: Rapid 5 min gradient for Tofersen ASO (sharp peak ~1.65 min); extended 15 min gradient for intact tRNAPhe (features ~9–10 min).
• LC systems and software: Thermo Scientific Vanquish Horizon (LC-UV) and Vanquish Neo UHPLC for LC–MS; Chromeleon 7.2.10 for LC-UV data.
• Mass spectrometry: Thermo Scientific Orbitrap Ascend Tribrid operated in negative-ion mode with Xcalibur 4.7; calibration with Pierce FlexMix. Typical MS settings included high-resolution full-scan (resolution 240,000), AGC target 4e5, scan ranges ~800–2800 m/z (ASO) and ~810–2500 m/z (tRNA). Data processing with Freestyle 1.8 and BioPharma Finder 5.3.

Main results and discussion

Particle morphology and column reproducibility:

• SEM comparison showed the SMP monodisperse particles have uniform size distribution versus conventional polydisperse particles. This uniformity supports improved column manufacturing consistency and lot-to-lot reproducibility, important for robust method development and transfer.

Tofersen ASO (20 nt) outcomes:

• Rapid LC–MS separation (5 min method) produced a sharp, symmetric ASO peak eluting at ~1.65 min, with a clear multiply charged ion envelope in the full-scan spectrum.
• Deconvolution revealed the intact neutral mass at 7123.170 Da representing the ASO. A minor species at 7252.322 Da was identified as a DBA adduct, consistent with the ion-pairing reagent in the mobile phase.
• This demonstrates fast intact-mass confirmation of short therapeutic oligonucleotides with strong MS response and confident mass assignment.

tRNAPhe (~75 nt) outcomes:

• The column resolved three major chromatographic features at ~9.2, 9.6 and 9.9 min under the longer gradient, enabling intact analysis of larger, structured RNA.
• Deconvoluted masses revealed a dominant intact tRNAPhe at 24610.450 Da, a demethylated variant at 24596.437 Da, and a minor sequence variant at 24625.454 Da (consistent with an A/U to G/C substitution). Thus, the platform supported detection of both chemical modifications and sequence variants in intact tRNA.

Overall performance observations:

• The monodisperse SMP reversed-phase medium demonstrated sufficient resolving power across a broad size range without requiring separate column chemistries for small oligonucleotides and larger RNAs.
• MS compatibility of the ion-pairing reagents and optimized source/scan parameters enabled high-resolution intact-mass assignment and straightforward identification of adducts and variants.

Benefits and practical applications

The platform provides several practical advantages:

• Unified workflow: single column chemistry capable of handling both short ASOs and larger RNAs reduces the need for multiple specialized columns and streamlines laboratory operations.
• Throughput: rapid methods (e.g., 5 min for ASO) support higher sample throughput for development and QC tasks.
• Intact-mass capability: high-resolution Orbitrap detection with careful ion-pairing conditions enables confident intact-mass confirmation and detection of modifications and adducts, valuable for impurity profiling and identity testing.
• Reproducibility and method transfer: monodisperse particle morphology improves manufacturing consistency and supports robust method transfer between labs and production lots.

Potential use cases include oligonucleotide therapeutic development, QC release testing, degradant and variant profiling, comparability studies, and workflows requiring rapid intact-mass confirmation.

Future trends and applications

Potential directions and broader opportunities include:

• Scaling the approach to even larger constructs (e.g., long mRNA fragments or full-length mRNA) and evaluating performance limits for higher molecular-weight species.
• Further optimization of ion-pairing chemistries, online desalting or trapping interfaces to reduce adduct formation and improve sensitivity for native or near-native analyses.
• Integration with automated sample prep and high-throughput MS acquisition workflows for screening and lot-release environments.
• Extension to hybrid or orthogonal separations (e.g., ion-exchange pre-fractionation or enzymatic digestion upstream of LC–MS) to support deeper characterization of complex therapeutic modalities.
• Regulatory adoption and method standardization as uniform, reproducible stationary phases become more widely available, enabling cross-laboratory comparability.

Conclusions

The 2.5 µm monodisperse supermacroporous SurePac Oligo RP MDi stationary phase demonstrated the capability to serve as an all-in-one reversed-phase LC–MS solution for nucleic acids spanning ~20 to ~75 nucleotides. The platform delivered fast, MS-compatible separations for a model ASO (Tofersen) and intact profiling of structured tRNA, including detection of chemical and sequence variants. Improved particle uniformity supports column reproducibility and robust method transfer, reducing the need for multiple specialized separation media in therapeutic nucleic acid analysis.

Used instrumentation

• Thermo Scientific Vanquish Neo UHPLC system (P/N VN-S10-A-01) for LC–MS runs.
• Thermo Scientific Vanquish Horizon UHPLC system for LC–UV experiments.
• Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer (P/N FSN06-10000), operated in negative-ion mode with Xcalibur 4.7; calibration with Pierce FlexMix.
• Data analysis: Thermo Scientific Freestyle 1.8 and BioPharma Finder 5.3; Chromeleon 7.2.10 for LC-UV data.
• Analytical column: Thermo Scientific SurePac Oligo RP MDi, 2.5 µm, 0.3 × 50 mm (P/N 43712050332).