(T1530-11-61) Advanced Characterization of COVID-19 mRNA Vaccines Using Litesizer DLS Instruments

Purpose: The primary objective of this study is to demonstrate the capabilities of Litesizer DLS instruments in analyzing the physical properties (particle size, surface charge, particle concentration) of mRNA vaccines. This research aims to demonstrate that dynamic and electrophoretic light scattering methods can provide critical insights into the behavior of lipid nanoparticle-formulated vaccines under different environmental conditions.

Methods: We utilized the Litesizer DLS instrument series to perform single-angle dynamic light scattering (DLS), multi-angle particle sizing (MAPS), electrophoretic light scattering (ELS) and particle concentration measurements on a commercial mRNA-based COVID-19 vaccine encapsulated in lipid nanoparticles. Samples were subjected to varying temperatures to simulate storage and handling conditions, observing changes in particle size distribution, concentration and zeta potential. The Litesizer DLS’s precision and analytical range enabled detailed characterization of these nanoparticle systems.

Results: The COVID-19 vaccine was first subjected to simulated cold-chained disruptions, consisting in a single freeze-thaw cycle, or heat treatment at +50 °C. Single-angle DLS confirmed the expected hydrodynamic diameter of lipid nanoparticles encapsulating the mRNA, which typically ranged between 80-100 nm under optimal storage conditions. We observed that the sample’s hydrodynamic diameter (HDD) is significantly increased in both heat-treated and, even more prominently, freeze-thawed samples, indicative of structural instability. Particle size changes in response to temperature were further investigated using multi-angle particle sizing (MAPS). Here we also observed a consistent and stable particle size distribution at temperatures up to +30 °C. At +40 °C, the lipid nanoparticles began showing signs of degradation and re-aggregation, as evidenced by an increase in particle size distribution bimodality. Particle sizes showed new peaks emerging at approximately 200 nm and 600 nm, suggesting the formation of fragmented particles and larger aggregates, respectively. Moreover, our MAPS-based particle concentration measurements detailed a marked increase in total particle concentration, which rose from approximately 1.4 E+12 particles/ml at +20 °C to 7.9 E+12 particles/ml at +70 °C. This uptick reflects not only the fragmentation of existing particles but also the dynamic nature of re-aggregation processes at elevated temperatures. Zeta potential, indicative of the surface charge of nanoparticles, was investigated using ELS. The optimally stored COVID-19 vaccine displays weakly negative zeta potential values. Interestingly, a slight but significant reduction of the absolute zeta potential is observed after heat-treatment, but not after freeze-thawing. These results underscore the sensitivity of lipid nanoparticles to thermal stress and highlight the Litesizer DLS’s capability to capture nuanced changes in a highly controlled environment. The detailed particle size and zeta potential analysis provided by the Litesizer DLS offers invaluable data for understanding the critical thresholds for vaccine storage and handling, ensuring vaccine efficacy and safety.

Conclusion: The study confirms the Litesizer DLS's utility as a powerful tool for the precise characterization of mRNA vaccines. The ability to accurately analyze particle size and concentration is crucial for ensuring the quality and efficacy of vaccines. Our findings emphasize the importance of maintaining strict temperature controls during the storage and handling of mRNA vaccines to prevent degradation and preserve vaccine efficacy.

Hydrodynamic diameter results for untreated, heattreated and freeze-thawed COVID-19 vaccine samples. Statistical significance between the different data sets, as determined by Student’s t-test, is shown on the relevant brackets.

Zeta potential results for untreated, heat-treated and freeze-thawed COVID-19 vaccine samples. Mean results from 7 consecutive measurements. Statistical significance between the different data sets, as determined by Student’s t-test, is shown on the relevant bracket.

Intensity-weighted particle size distribution at starting temperature of 20 °C and after increasing the temperature up to 70°C.