A novel monodisperse supermacroporous reversed-phase platform for comprehensive nucleic acid analysis across a broad size range

Significance of the topic

Comprehensive, high-resolution analytical methods for nucleic acid therapeutics are essential across discovery, process development, stability testing and quality control. Therapeutic modalities span from short siRNA and oligonucleotides to long mRNA constructs, creating a demand for chromatographic platforms that provide consistent separation performance across wide size and structural heterogeneity. A single robust reversed-phase platform capable of resolving small oligonucleotides, intermediate sgRNAs/tRNAs and large intact mRNA reduces method complexity, improves throughput and simplifies method transfer between laboratories and production sites.

Objectives and study overview

This work evaluates a novel 2.5 μm monodisperse supermacroporous (SMP) reversed-phase polymeric stationary phase (SurePac Oligo RP MDi) as an all-in-one platform for nucleic acid analysis. The aim was to demonstrate that the monodisperse SMP material provides high resolving power, low carryover and reproducible performance across a broad molecular weight range (short siRNA 20–25 bp through long mRNA up to ~2,000 nt) and to compare its performance to other commercial columns and media.

Methodology and used instrumentation

• Stationary phase: 2.5 μm monodisperse supermacroporous polymeric resin, hydrophobic-coated stainless-steel inert hardware (SurePac Oligo RP MDi).
• Columns evaluated: SurePac Oligo RP MDi (2.5 μm, 2.1 × 50 mm and 0.3 × 50 mm), DNAPac RP (4 μm, 2.1 × 50 mm) and multiple vendor C18 columns (various particle sizes and pore diameters).
• UHPLC systems: Thermo Scientific Vanquish Horizon for LC-UV experiments and Vanquish Neo for LC–MS experiments.
• Mass spectrometry: Thermo Scientific Orbitrap Ascend Tribrid high-resolution MS operated in negative ion mode for intact RNA profiling; data processed with Xcalibur, Freestyle and BioPharma Finder.
• Data system: Thermo Scientific Chromeleon 7.2.10 for LC-UV acquisition/analysis.
• Mobile-phase compatibility: demonstrated with TEAA/acetonitrile, HAA/acetonitrile and volatile HFIP/DBA/methanol ion-pairing systems to enable LC–MS workflows.
• Samples tested: 21 bp siRNA, custom 8-combo ssDNA mix (12–40mer), sgRNA (~100 nt), tRNAPHE (~75 nt, ~25 kDa), and an uncapped 1960 nt mRNA transcript.

Main results and discussion

• Particle uniformity and packing reproducibility: SEM comparison showed monodisperse particles yield narrower particle size distributions than polydisperse media, which translated into more uniform flow paths, reduced packing heterogeneity and improved lot-to-lot and column-to-column reproducibility.
• Resolution across size range: The SurePac Oligo RP MDi column delivered sharper peak shapes and improved separation of closely eluting impurities for short siRNA samples, resolving more minor variants and providing better antisense/sense separation than comparator columns.
• Intermediate-size performance: For sgRNA (~100 nt) the monodisperse SMP column produced a more detailed impurity profile and better peak shape, likely due to favorable pore accessibility and selectivity compared with alternative stationary phases.
• Large mRNA analysis: For a 1960 nt mRNA transcript the SMP column exhibited improved peak shape and detected an early-eluting proximal species that other columns either poorly resolved or showed severe tailing on, consistent with better pore accessibility and effective surface area for large molecules.
• LC–MS intact RNA profiling: Under HFIP/DBA ion-pairing conditions, intact tRNAPHE (~25 kDa) analysis detected ten reproducible molecular states above a 5% threshold across four replicates. Monoisotopic mass precision was ≤1 ppm for major species, with the dominant state showing 0.07 ppm CV. Closely related variants differing by 14–16 Da were reproducibly resolved and assigned.
• Carryover and throughput: Low carryover was observed (0.48% following a concentrated mRNA injection), an important metric for high-throughput impurity analysis and accurate quantitation of low-level species.
• Reproducibility: Using an 8-combo ssDNA method, three different production lots of the SurePac Oligo RP MDi column demonstrated consistent retention times and peak widths, supporting robust method transfer.

Benefits and practical applications

• Single-platform capability: The monodisperse SMP column functions across a wide nucleic acid size spectrum, minimizing the need to maintain multiple specialized columns for different analyte sizes.
• Enhanced impurity detection: Improved peak shape and resolution enable finer detection and quantitation of process- and stability-related impurities, which benefits process development, formulation stability studies and QC release testing.
• LC–MS compatibility: Demonstrated compatibility with volatile ion-pairing mobile phases and high-resolution MS workflows enables intact mass analysis and detection of modification heterogeneity.
• Operational robustness: Low carryover, strong lot-to-lot reproducibility and inert hardware support high-throughput analytical labs and routine QC environments.

Used instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system (LC-UV experiments).
• Thermo Scientific Vanquish Neo UHPLC system (P/N VN-S10-A-01) for LC–MS experiments.
• Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer (P/N FSN06-10000) with Thermo Scientific Xcalibur 4.7 software; calibration using Pierce FlexMix.
• Data software: Chromeleon 7.2.10 (LC-UV), Freestyle 1.8 and BioPharma Finder 5.3 (MS data analysis).
• Columns: Thermo Scientific SurePac Oligo RP MDi (2.5 μm, 2.1 × 50 mm, P/N 43712-052132; and 0.3 × 50 mm, P/N 43712-050332); Thermo Scientific DNAPac RP (4 μm, 2.1 × 50 mm, P/N 088924); plus vendor C18 columns used for comparison.

Future trends and potential applications

• Broader adoption in mRNA and oligonucleotide therapeutic QC workflows as the demand for unified analytical platforms grows.
• Further optimization for even longer or heavily modified RNAs and integration with native or top-down MS approaches to enable site-specific modification mapping in combination with orthogonal enzymatic workflows.
• Continued stationary-phase engineering to refine pore architectures for improved accessibility of very large biopolymers while retaining high resolving power for short oligonucleotides.
• Automation and method standardization across multi-site manufacturing to facilitate regulatory submissions and lot-release testing for nucleic acid therapeutics.

Conclusion

The 2.5 μm monodisperse supermacroporous SurePac Oligo RP MDi stationary phase demonstrates that a single reversed-phase platform can deliver high-resolution separations across a broad nucleic acid size range, from short siRNA and oligonucleotides to intact mRNA. Key advantages include improved peak shape and impurity resolution, excellent lot-to-lot reproducibility, LC–MS compatibility with volatile ion-pairing systems, low carryover and reliable intact-mass performance. These attributes make the platform a practical option for streamlining analytical workflows in research, development and QC for nucleic acid therapeutics.

References

Ma K., Ross R., Bechler S., Mitter C., De Pra M., Lin S. A novel monodisperse supermacroporous reversed-phase platform for comprehensive nucleic acid analysis across a broad size range. Thermo Fisher Scientific. PO004767 EN 0626, 2026.