Analysis of mRNA-Lipid Nanoparticles in Human Plasma using SEC-Static Dual-Angle Light Scattering and Refractive Index Detection

Importance of the Topic

Messenger RNA lipid nanoparticles (mRNA-LNPs) have emerged as a key delivery vehicle for nucleic acid therapeutics, exemplified by COVID-19 vaccines. Their behavior in physiological fluids strongly influences drug stability, pharmacokinetics and safety. Assessing mRNA-LNP integrity and degradation in complex media such as human plasma is therefore critical for guiding formulation development, storage conditions and regulatory compliance.

Objectives and Study Overview

This application note details a study using size exclusion chromatography (SEC) combined with online static dual-angle light scattering (LS) and differential refractive index (RI) detection to monitor mRNA-LNP interactions with human plasma. The goals were to demonstrate high-resolution separation of nanoparticles from plasma components, to quantify changes in hydrodynamic radius (Rh) and molecular weight (Mw) in real time, and to compare reaction kinetics in plasma versus isolated serum albumin.

Methodology and Instrumentation

mRNA-LNPs were prepared using a composition matching Pfizer/BioNTech’s Comirnaty formulation via microfluidic mixing of aqueous mRNA solution and ethanol-dissolved lipids. After ultracentrifugation and buffer exchange into saline, particles (~2.2 mg/mL lipid) were characterized by bulk dynamic light scattering (DLS) and by SEC-multidetector analysis. Human plasma powder was reconstituted, clarified by centrifugation and filtration. mRNA-LNPs were diluted into plasma or 40 mg/mL serum albumin solution and injected at 15-minute intervals for up to 120 minutes.

Instrumentation Used

• Agilent 1290 Infinity II Bio LC with quaternary pump (isocratic mode)
• Agilent 1260 Infinity II Bio-SEC multidetector system (dual-angle LS at 15°/90° and dynamic LS)
• Agilent 1290 Infinity II diode array detector (for system calibration)
• Agilent 1290 Infinity II refractive index detector
• Two PL aquagel-OH SEC columns (4.6×250 mm) with 500 kDa and 10 MDa pore size, used in series for enhanced resolution

Main Results and Discussion

• High resolution of mRNA-LNPs from plasma proteins was achieved only with the dual-column SEC setup; single-column runs produced coelution and inaccurate concentration estimates.
• Online DLS and static LS enabled real-time measurement of Rh, Mw and radius of gyration (Rg), revealing an initial Rh of ~32 nm and Mw ~40 MDa consistent with monodisperse spherical particles (Rg/Rh ≈0.9).
• In human plasma, the mRNA-LNP peak area and Mw decreased linearly over ~120 minutes (zero-order kinetics), while Rh dropped to ~25 nm, indicating nanoparticle degradation without significant protein adsorption.
• In contrast, incubation with serum albumin led to a ~25% increase in Mw within 30 minutes but negligible change in Rh, consistent with a reversible protein-coating rather than particle breakdown.

Benefits and Practical Applications

SEC coupled with multidetector LS and RI provides unbiased profiling of all particulate species in complex matrices, surpassing bulk DLS in sensitivity and avoiding matrix interference. This approach facilitates stability testing, quality control of nanomedicines, optimization of formulation strategies and compliance with regulatory guidance on nanomaterial–plasma interactions.

Future Trends and Opportunities

Advances may include integrating mass spectrometry or multi-wavelength detectors for compositional analysis, employing novel column materials for broader size ranges, and coupling microfluidic reactors for automated on-line incubation. Expanding applications to other nanocarriers, personalized dosing studies and in vitro–in vivo correlation models will further enhance nanoparticle characterization.

Conclusion

The combination of high-resolution SEC and multidetector light scattering offers a powerful platform for monitoring mRNA-LNP stability and interactions in plasma. It delivers orthogonal size and weight information, discriminates degradation from protein adsorption, and supports streamlined development and regulatory assessment of nanoparticle-based therapeutics.

References

1. Tenchov R; Bird R; Curtze AE; Zhou Q. Lipid Nanoparticles–From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano 2021.
2. U.S. Food and Drug Administration. Guidance for Industry: Drug Products, Including Biological Products, that Contain Nanomaterials. April 2022.