Accurate Particles Characterization in Lipid Nanoparticle Therapies

Importance of Lipid Nanoparticle Particle Characterization

Lipid nanoparticles (LNPs) are a critical delivery platform for mRNA therapies and vaccines due to their capacity to encapsulate genetic payloads, low immunogenicity, and scalable production. Ensuring the physical stability of LNP formulations requires reliable detection of subvisible particles (SVPs) that may lead to aggregation, reduced efficacy, or safety concerns in injectable products.

Objectives and Study Overview

This application note demonstrates the use of the Aura GT System with Backgrounded Membrane Imaging (BMI) and Fluorescence Membrane Microscopy (FMM) to quantify and characterize SVPs in LNP formulations. Two model LNP samples, designated A and B, were assessed under various stress conditions and buffer compositions to evaluate the sensitivity, throughput, and low-sample-volume capability of the method.

Methodology and Instrumentation

Two LNP samples containing ionizable lipids, cholesterol, DSPC, PEG-DMG or SM-102, and RNA (45 µg/mL) were prepared, with mean diameters of 110 nm (Sample A) and 140 nm (Sample B) by DLS. Forced degradation included:

• Thermal stress: 80 °C for 2 h
• Freeze-thaw cycles: three rounds between –80 °C and 23 °C over 6 h
• Formulation buffers: acetate (pH 4.8), PBS, glycine/arginine (pH 10.6)

SVP analysis employed 25 µL sample volumes per well on a black membrane plate using BMI for size and count, and FMM with SYBR Gold staining to detect nucleic acid–coated particles. The Aura GT System enabled rapid imaging and quantitation across ≥2 µm, ≥10 µm, and ≥25 µm size ranges.

Key Results and Discussion

Both LNP samples exhibited high SVP counts (>2×10^6 particles/mL for ≥2 µm) in unstressed conditions. Freeze-thaw stress increased counts by 47% for Sample A and 25% for Sample B, while thermal stress reduced SVP formation 5- to 10-fold across both samples. Sample A tended to produce larger SVPs (≥10 µm) than Sample B in all conditions. SYBR Gold fluorescence revealed significant nucleic acid exposure in freeze-thaw stressed particles but not in unstressed samples, indicating vesicle rupture and payload leakage. Buffer type had minimal effect on SVP levels, showing robust formulation stability across pH and ionic conditions. Filtration experiments demonstrated that two successive 0.2 µm membrane passes were required to clear SVPs, with reappearance of particles indicating concentration-dependent aggregation post-filtration.

Benefits and Practical Applications

• High sensitivity to low-volume samples (25 µL) accelerates screening during formulation development.
• BMI and FMM combination identifies both physical particles and nucleic acid release, informing stability and integrity assessments.
• Compliance with USP 787/788 requirements for SVP testing in colloidal suspensions.
• Applicable to quality control, process monitoring, and release testing of LNP-based therapeutics.

Future Trends and Opportunities

Advancements may include integration with automated sample handling, real-time monitoring of SVP formation during manufacturing, machine-learning–driven image analysis for particle classification, and expansion to emerging LNP designs with multifunctional payloads. Regulatory acceptance of advanced imaging methods could further streamline LNP product development.

Conclusion

The Aura GT System provides rapid, accurate, and low-volume quantitation of subvisible particles in lipid nanoparticle formulations. Its combined BMI and fluorescence capabilities address limitations of conventional light-obscuration methods and support comprehensive stability and release testing of complex LNP therapeutics.

References

• Xu L, Wang X, Liu Y, Yang G, Falconer RJ, Zhao C. Lipid Nanoparticles for Drug Delivery. Adv NanoBiomed Res. 2022;2:2100109.
• Ball RL, Bajaj P, Whitehead KA. Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization. Int J Nanomedicine. 2016;12:305–315.
• Trenkenschuh E, Savšek U, Friess W. Formulation, process, and storage strategies for lyophilizates of lipophilic nanoparticulate systems. Int J Pharm. 2021;606:120929.