Comparison of MALS-Based Analyses of Large mRNA Separated on Widepore SEC Columns

Significance of the topic

Messenger RNA therapeutics are rapidly emerging across various clinical areas, driving a need for precise size and mass analysis of large mRNA molecules. Coupling size exclusion chromatography with multi-angle light scattering enables absolute characterization of macromolecules, supporting method development for next generation drugs.

Objectives and study overview

This study compares two widepore SEC columns—GTxResolve Premier SEC 1000 Å 3 µm and a manufacturer A 1000 Å 2.7 µm column—in their ability to separate and support robust MALS analysis of large mRNA samples under fast measurement and minimal sample conditions.

Methodology and instrumentation

A BSA standard and Cas9 mRNA sample were analyzed by SEC-MALS using an ACQUITY Premier quaternary system coupled to a Wyatt DAWN 18-angle light scattering detector and UV absorbance at 260 nm. Columns were equilibrated with 0.2 µm filtered PBS. Instrument constants were determined via BSA injections and system suitability validated prior to mRNA analysis. 1 µg of mRNA was injected for performance evaluation.

Main results and discussion

Noise evaluation after 20 column volumes of flushing revealed that the GTxResolve column displayed almost four times lower MALS background noise compared to the manufacturer A column, ensuring reliable low-level sample measurements. System suitability with BSA showed sharper separation and higher precision for mass determinations on the GTxResolve column. Analysis of 1 µg Cas9 mRNA achieved single-digit precision for monomer and dimer molecular masses and radius of gyration within 15% of predicted values only on the GTxResolve column; the manufacturer A column yielded less accurate results.

Benefits and practical applications

The GTxResolve Premier SEC 1000 Å column enables robust SEC-MALS workflows with reduced noise and lower sample requirements, facilitating high-throughput screening of mRNA therapeutics and supporting standardized validation procedures.

Future trends and potential uses

As nucleic acid therapeutics expand, advances in column pore architecture, surface chemistries, and mobile phases will further enhance SEC-MALS performance. Integration with complementary detectors and development of protocols for new modalities will support personalized medicine and complex biologic characterization.

Conclusion

This comparison highlights that column design features such as broad pore size distribution and low particle shedding are critical for fit-for-purpose SEC-MALS analysis of large mRNA. The GTxResolve Premier SEC 1000 Å column demonstrated superior noise performance and reliable molecular characterization with limited sample amounts.

References

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2. Waters/Wyatt Technical Note TN3506 Molecular Standards for Determining System Constants and Validating SEC-MALS-IV and FFF MALS Systems. 2018.
3. Waters Application Note 720008669 Optimized Method Conditions for mRNA Characterization by SEC-MALS With GTxResolve Premier SEC 1000 Å 3 µm Columns. 2025.
4. GenScript Product Information Sheet for SpCas9 Methylpseudouridine mRNA, accessed 02/01/2025.
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