Optimized Method Conditions for mRNA Characterization by SEC-MALS With GTxResolve™ Premier SEC 1000 Å 3 μm Columns

Importance of the Topic

Messenger RNA therapies require rigorous characterization of molecular attributes to guarantee product safety, efficacy and consistency. Size-exclusion chromatography combined with multi-angle light scattering (SEC-MALS) provides absolute measurements of molar mass, size, aggregation and conformation, critical for quality control of mRNA drug substances.

Objectives and Study Overview

This study evaluated GTxResolve Premier SEC 1000 Å 3 µm columns for SEC-MALS analysis of mRNA and a dsDNA ladder. A systematic design of experiments was conducted to optimize mobile phase composition, ionic strength and temperature to achieve high resolution separation and accurate quantitation of monomer, aggregate and fragment species. The optimized method was applied to Cas9, firefly luciferase and erythropoietin mRNA samples.

Methodology

A two-level factorial screening followed by full factorial DoE investigated the effects of buffer type, chaotropic versus kosmotropic additives, ionic strength and column temperature on separation performance. Secondary interactions were minimized by selecting 50 mM Tris pH 7.5 with 250 mM ammonium chloride and operating at 40 °C. SEC-MALS runs were performed isocratically at 0.30 mL/min, coupling UV absorption, multi-angle light scattering and refractive index detection to obtain absolute molar mass, radius of gyration, hydrodynamic radius and extinction coefficients.

Instrumentation Used

• LC System Arc Premier with Quaternary Solvent Manager and Flow Through Needle Sample Manager
• Columns GTxResolve Premier SEC 1000 Å, 3 µm, 7.8 x 300 mm and 4.6 x 150 mm
• Detectors Wyatt DAWN MALS with embedded WyattQELS dynamic light scattering, Arc Premier PDA at 260 and 280 nm, Wyatt Optilab dRI
• Software HPLC CONNECT for system control and ASTRA for data acquisition and analysis

Main Results and Discussion

• Design of experiments identified ammonium salts, elevated ionic strength and higher temperature as key factors improving resolution and recovery while reducing high molecular weight species.
• The dsDNA ladder was fully resolved into 17 species with measured molar masses matching expected values (slope 1.03 in regression).
• Cas9 mRNA aggregates decreased from about 33 percent mass fraction in the native sample to 2 percent after denaturation and reannealing, and monomer molar mass aligned within 1 percent of theoretical.
• Firefly luciferase and erythropoietin mRNA measurements yielded molar masses, extinction coefficients and A260/A280 ratios consistent with predicted values, demonstrating method applicability across mRNA types.
• Conformational analysis by Rg versus molar mass showed mRNA behaves as a compact coil while dsDNA adopts an extended rod structure, highlighting the limitations of calibration curve approaches.

Benefits and Practical Applications

• Absolute quantitation of mRNA molar mass, size, aggregation and conformation in a single run.
• High resolution separation of monomer, aggregates and fragments using optimized mobile phase.
• Low background noise and minimal sample interaction provided by hydrophilic surface-modified columns.
• Efficient quality control workflow for mRNA therapeutics in research and commercial manufacturing.

Future Trends and Opportunities

Integration of automated method development, expansion to longer and modified RNA constructs, high-throughput SEC-MALS workflows, and coupling with orthogonal techniques such as mass spectrometry and capillary electrophoresis will further enhance characterization of complex nucleic acid therapeutics.

Conclusion

Optimized SEC-MALS using GTxResolve Premier SEC 1000 Å 3 µm columns delivers robust, reproducible and absolute measurement of critical quality attributes of mRNA drug substances, supporting the development and quality control of advanced gene therapy and vaccine products.

References

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