ASGTC: mRNA/LNP Multiattribute Quantitation of Payload(s), Size and Heterogeneity With Size Exclusion Chromatography Coupled to Multiangle Light Scattering

Significance of the Topic

Understanding the physical and compositional properties of mRNA/lipid nanoparticles is vital for consistent manufacturing, regulatory compliance and clinical performance of RNA-based pharmaceuticals. SEC-MALS offers a platform to evaluate size, molecular weight and payload integrity in a streamlined workflow.

Objectives and Study Overview

This work presents two complementary SEC approaches for mRNA/LNP analysis:

• Native size-based fractionation of intact LNPs with UV-MALS-dRI detection to characterize size distribution and heterogeneity.
• Denaturing SEC with a disruptive mobile phase to achieve online deformulation and accurate quantification of multiple RNA payloads.

Methodology and Instrumentation

Key experimental details include:

• LNP samples: Commercial vaccines (Spikevax, Comirnaty) and in-house formulations carrying dual payloads (mRNA and gRNA).
• Native SEC: GTxResolve Premier SEC 1000 Å, 3 µm, PEO bonded column with 1X PBS mobile phase at 25 °C, flow rate 0.3 mL/min.
• Denaturing SEC: GTxResolve Premier BEH SEC 450 Å, 2.5 µm column with 1X PBS, 20% isopropanol and 0.2% SDS, 40 °C, flow rates of 0.25–0.5 mL/min.
• Detection: UV at 260 nm, multi-angle light scattering, refractive index and dynamic light scattering modules (Waters ACQUITY systems and Wyatt detectors).

Main Results and Discussion

• Native SEC-MALS achieved fractionation of intact LNPs (40–100+ nm) with recoveries of 50–60%, enabling size and polydispersity profiling without prior sample preparation.
• Denaturing SEC conditions yielded complete online deformulation of all tested LNPs, releasing mRNA and gRNA payloads and eliminating light scattering contributions.
• Optimized mobile phase allowed quantification of dual payloads in a 10 min method (extendable to 5 min) with recovery reproducibility below 5% and limits of quantification around 7 ng.
• DLS data confirmed narrow hydrodynamic radius distributions, and UV absorbance ratios (A260/A230) validated complete particle disruption.

Benefits and Practical Applications of the Method

• High-throughput analysis combining size, heterogeneity and payload content in a single workflow.
• Elimination of laborious offline extraction steps for multi-payload quantification.
• Robust approach applicable to various LNP formulations, including commercial vaccines.
• Improved method reproducibility and reduced development time compared to traditional RPLC or electrophoretic techniques.

Future Trends and Applications

• Further reduction of analysis time through column and flow optimization.
• Adoption of novel wide-pore stationary phases for broader nanoparticle characterization.
• Integration with automated sample handling and data analysis pipelines.
• Expansion to other nanocarrier systems and complex multi-component therapeutics.

Conclusion

SEC coupled with MALS and denaturing mobile phases provides a versatile, high-throughput platform for simultaneous assessment of mRNA/LNP size, heterogeneity and payload quantification. The described methods deliver reproducible results and streamline quality control workflows for RNA nanoparticle-based products.

References

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2. Jia X, Pennington J, et al. Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations. J Chromatogr B. 2021;1186:123015.
3. Lokras A, Foged C, et al. Simultaneous quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems. Int J Pharm. 2022;626:122171.