SEC-MALS Method for Characterizing mRNA Biophysical Attributes

Significance of the Topic

The development of mRNA therapeutics has created a pressing need for robust analytical methods to define molecular weight, aggregate content, and conformational parameters. Accurate biophysical characterization guides formulation strategies, assesses stability, and supports regulatory submissions for vaccines, immuno-oncology agents, and protein replacement therapies.

Objectives and Study Overview

This study presents a size-exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering detectors (SEC-MALS/DLS) to quantify key properties of mRNA constructs. Two model transcripts encoding human erythropoietin (EPO) and firefly luciferase (fLuc) are analyzed to demonstrate simultaneous determination of molecular weight, percentage of aggregates, radius of gyration, hydrodynamic radius, and Rg/Rh ratio in a single run.

Methodology

In vitro transcription generated purified EPO and fLuc mRNA samples, which were quantified by UV absorbance. Separation was performed on an HPLC platform using a WTC050S5 SEC column with phosphate-buffered saline (pH 6.8) at 0.8 mL/min. Eluent streams were monitored by UV absorbance at 260 nm for mRNA and 280 nm for any protein impurities.

Used Instrumentation

• HPLC system with WTC050S5 SEC column
• Variable wavelength UV detector
• DAWN multi-angle light scattering detector equipped with an embedded DLS module
• Optilab differential refractive index detector
• ASTRA software for instrument control, data acquisition, and analysis

Main Results and Discussion

The SEC-MALS/DLS method resolved a primary monomer peak and minor high-molecular‐weight aggregates for both mRNA samples. MALS/RID analysis yielded molecular weights of 272 ± 1 kDa for EPO mRNA and 622 ± 1 kDa for fLuc mRNA, matching their theoretical values. Aggregate percentages were 4.8% and 2.6%, respectively. Radius of gyration values of 15 ± 1 nm and 20 ± 1 nm and hydrodynamic radii of 12 ± 1 nm and 17 ± 1 nm indicate an Rg/Rh ratio around 1.2, consistent with a random coil conformation. These measurements were achieved in under 15 minutes per run, underscoring throughput and reproducibility.

Benefits and Practical Applications

The SEC-MALS/DLS approach offers absolute molecular weight determination independent of calibration standards, avoiding the overestimation common with protein-based size calibrants. Simultaneous measurement of size, conformation, and aggregation in a single assay streamlines characterization workflows. This capability supports formulation optimization, stability testing, and quality control during mRNA drug development.

Future Trends and Applications

Advancements may include integration with high-throughput autosamplers and microfluidic SEC systems to accelerate screening of formulation variants. Expanded use of artificial intelligence for data interpretation could reveal subtle conformational changes. Coupling with orthogonal techniques such as small-angle X-ray scattering or mass photometry will further enrich structural understanding. The platform can be adapted to emerging RNA modalities, including circular RNA and self-amplifying constructs, and to studies of lipid nanoparticle encapsulation effects.

Conclusion

The SEC-MALS/DLS method provides a comprehensive, rapid, and reliable tool for characterizing mRNA biophysical attributes. Its ability to measure molecular weight, size, conformation, and aggregation concurrently makes it essential for advancing mRNA therapeutic research and development.

References

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