Comprehensive analysis of nucleic acids across a wide size range using a monodisperse reversed-phase particle platform

Significance of the topic

The development and regulatory advancement of nucleic acid therapeutics require robust analytical workflows capable of resolving molecules from short synthetic oligonucleotides (tens of nucleotides) up to long mRNA constructs (>1,000 nt). A single chromatographic platform that provides high resolution, reproducible performance, low carryover, and compatibility with LC–MS accelerates method development, QC, process monitoring, stability testing, and method transfer across discovery and manufacturing pipelines.

Goals and overview of the study

This application note evaluated the Thermo Scientific SurePac Oligo RP MDi reversed-phase column (2.5 µm monodisperse supermacroporous polymer particles) as a unified chromatographic solution for nucleic acids spanning ~10 to >1,000 nucleotides. The column’s performance was compared versus DNAPac RP (polymeric 4 µm) and several commercially available silica-based reversed-phase columns across sample classes including short ssDNA mixes, siRNA, sgRNA, ~100 nt oligonucleotides, a dsDNA ladder (72–1,353 bp), tRNAPhe, and ~1,960 nt mRNA. The work also assessed compatibility with different ion-pair mobile phases and LC–MS workflows.

Methodology

The study used targeted chromatographic methods optimized per sample class and evaluated chromatographic metrics (resolution, peak shape, retention window, backpressure, carryover) and MS-based intact mass analysis under volatile ion-pairing conditions. Mobile phases tested included TEAA/ACN (traditional acetate-based ion pair), hexylammonium acetate (HAA)/ACN, and volatile HFIP/DBA with methanol for MS compatibility. Samples spanned: an 8-component ssDNA ladder (12–40 mer), 21 bp siRNA control, 98/99/100-mer ssDNA mix, ~100 nt sgRNA, phiX174 dsDNA digest ladder, tRNAPhe, and an in-house synthesized ~1,960 nt mRNA. Chromatographic formats included 2.1 × 50 mm and 0.3 × 50 mm SurePac columns and comparable vendor columns; temperatures, gradients and flow rates were adjusted for each evaluation.

Used instrumentation

• Thermo Scientific Vanquish Neo UHPLC and Vanquish Horizon UHPLC platforms (system base, binary pump, column compartment, active pre-heater, post-column cooler, split sampler).
• Vanquish Diode Array Detector HL with LightPipe flow cell for UV detection.
• Thermo Scientific Orbitrap Ascend Tribrid Mass Spectrometer for high-resolution intact mass analysis (negative ion mode), with Xcalibur control and FreeStyle/BioPharma Finder data analysis.
• Chromeleon CDS for data acquisition and processing.

Main results and discussion

• Short oligonucleotides (12–40 mer, 21 bp siRNA): The SurePac Oligo RP MDi column produced equivalent or sharper peaks and better resolution of closely eluting impurities compared to DNAPac RP and multiple vendor reversed-phase columns. It provided broader peak spread across the gradient window and resolved more minor impurity peaks for the siRNA control, including clear antisense/sense separation.
• Mid-size oligonucleotides (~98–100 nt and sgRNA ~100 nt): The MDi column achieved baseline or near-baseline separation of 98-, 99-, and 100-mer ssDNA, outperforming other columns which showed reduced or no separation. The column delivered the most detailed impurity profiles for sgRNA, with improved peak shape and selectivity attributed to accessible pore structure across the particle.
• Large nucleic acids (dsDNA ladder and ~1,960 nt mRNA): The SurePac Oligo RP MDi column matched or exceeded DNAPac RP in accessibility for large analytes while offering superior resolving power. Other evaluated columns struggled above ~310 bp. mRNA analysis showed improved peak shapes and detection of proximal early-eluting species; carryover after high-concentration mRNA injection was low (~1%).
• Mobile phase flexibility: The stationary phase performed robustly with TEAA/ACN, HAA/ACN, and volatile HFIP/DBA/methanol systems. HAA/ACN preserved sharp peaks and good selectivity; HFIP/DBA enabled direct LC–MS coupling with maintained peak symmetry and impurity resolution.
• LC–MS intact RNA profiling: Using HFIP/DBA conditions and high-resolution Orbitrap analysis, intact tRNAPhe (~25 kDa) produced reproducible detection of ~10 molecular states above 5% relative abundance. Monoisotopic mass accuracy was ≤1 ppm across replicates and injection-to-injection mass CVs were extremely low (e.g., dominant intact state 0.07 ppm CV), demonstrating robust mass reconstruction and variant assignment.
• Operational advantages: The monodisperse SMP particles gave consistent particle size distribution, translating into improved lot-to-lot and column-to-column reproducibility, lower backpressure than several silica-based alternatives, and reliable retention time and peak width reproducibility across lots.

Benefits and practical applications

• Single-platform solution: The SurePac Oligo RP MDi column enables characterization across a broad size range (short oligos to long mRNA) reducing the need to maintain multiple column types for different assays.
• Improved impurity and truncated-species detection: High resolving power for single-nucleotide differences and minor variants supports purity assessment, process development monitoring, and stability studies.
• LC–MS compatibility: Performance with volatile ion-pair reagents enables direct coupling to high-resolution MS for identity confirmation and intact mass profiling of RNA species.
• Operational robustness: Lower operating backpressure, low carryover, and demonstrated lot-to-lot reproducibility facilitate routine QC deployment and method transfer.

Future trends and opportunities

• Unified chromatographic platforms: Demand for single-column solutions will grow as therapeutic oligonucleotides and mRNA constructs diversify in length and chemistry, motivating broader adoption of stationary phases that balance pore accessibility and resolving power.
• MS-driven characterization: Greater reliance on volatile ion-pairing chemistries and high-resolution MS for intact mass and impurity identification will increase, pushing further optimization of MS-compatible mobile phases and source/interface technologies.
• High-throughput and automated workflows: Low backpressure and robust lot reproducibility support higher flow or multiplexed formats for QC labs; integration with automated sample prep and data pipelines will accelerate screening and release testing.
• Regulatory and standardization needs: As modalities progress toward commercialization, standardized methods and column performance expectations will be important for method transfer, cross-site reproducibility, and regulatory submissions.

Conclusion

The SurePac Oligo RP MDi column, built on 2.5 µm monodisperse SMP polymer particles, delivers a practical, high-resolution reversed-phase solution for nucleic acid analysis across a wide size range. It combines improved separation of short oligonucleotides, strong performance for mid- and large-size RNA/DNA, compatibility with multiple ion-pair mobile phases (including MS‑compatible systems), low carryover, and reliable lot-to-lot reproducibility. These characteristics make it suitable for analytical needs spanning discovery, process development, QC, and LC–MS-based characterization workflows.