Deformulating Size Exclusion Chromatography for LNP Payload Quantitation

Significance of the Topic

Lipid nanoparticles (LNPs) are central to modern gene therapies and vaccines, delivering mRNA payloads to target cells. Reliable quantification and integrity assessment of the nucleic acid cargo within LNPs are critical for ensuring safety and efficacy during development and manufacturing. Traditional UV absorption methods are confounded by scattering from intact LNPs, and common denaturing protocols can require laborious sample preparation or may be incompatible with chromatographic separation.

Study Objectives and Overview

This study aimed to develop an inline deformulating size exclusion chromatography (SEC) method using a GTxResolve Premier BEH SEC 450 Å column coupled with UV detection to separate and quantify mRNA payloads from intact LNPs without offline sample preparation. By incorporating specific mobile phase additives, the method seeks to achieve rapid and complete disruption of LNPs directly within the LC system.

Methodology and Instrumentation

• Instrumentation:
  • ACQUITY UPLC H-Class PLUS Bio System with quaternary pump
  • ACQUITY UPLC Tunable UV Detector (260 nm and 230 nm)
  • GTxResolve Premier BEH SEC 450 Å Column (4.6 × 150 mm, 2.5 µm)
• Chromatographic Conditions:
  • Mobile phase: 1×PBS (pH 7.4) with 20% isopropanol and 0.2% sodium dodecyl sulfate, filtered
  • Flow rate: 0.25–0.5 mL/min
  • Column temperature: 40 °C; sample temperature: 6 °C
  • Injection volume: 0.1–1.0 µL
• Samples:
  • Commercial COVID-19 mRNA vaccines (Spikevax™, Comirnaty™) and model LNPs containing firefly luciferase or Cas9 mRNA
  • Reference mRNA isolated by alcohol precipitation for method validation

Main Results and Discussion

• Native SEC under physiological conditions showed incomplete LNP disruption and UV scattering interference.
• Offline Triton X-100 treatment failed to fully release mRNA, leaving residual LNP peaks and absorption artifacts.
• A deformulating mobile phase containing 0.2% SDS and 20% isopropanol achieved complete inline LNP disruption, as evidenced by A260/230 ratios nearing 2.0.
• The method separated mRNA payloads of different sizes (∼5000 nt vs. ∼2000 nt), enabling distinction of multiple payloads such as Cas9 mRNA and guide RNA in gene editing LNPs.
• Quantitative recovery was confirmed through consistent UV responses and linear detector calibration across LNP concentrations.

Benefits and Practical Applications

• Fast analysis (<10 minutes) with no offline sample preparation.
• Robust and reproducible quantification of mRNA payload integrity and concentration.
• Cost-effective and easily deployable platform for vaccine and gene editing LNP quality control.
• Suitable for multi-payload formulations and potential extension to other nanoparticle modalities.

Future Trends and Applications

LNP-based therapies are rapidly evolving, with growing complexity in multi-component drug products. Inline deformulating SEC-UV methods could be expanded to protein-based nanoparticles, adeno-associated virus vectors, and other advanced delivery systems. Future integration with multi-angle light scattering or mass spectrometry detection may further enhance characterization capabilities and support high-throughput process development and quality assurance workflows.

Conclusion

The developed inline deformulating SEC-UV assay using a GTxResolve Premier BEH SEC 450 Å column and a mobile phase with SDS and isopropanol provides a rapid, universal, and robust tool for releasing, separating, and quantifying mRNA payloads from LNP carriers without additional sample preparation. This platform method supports the analytical needs of emerging mRNA and gene editing therapeutics by enabling accurate assessment of payload content and integrity.

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