(M0930-09-56) Reliable Sizing of Lipid Nanoparticles in Concentrated Formulations Using Advanced Dynamic Light Scattering

Purpose: Understanding the impact of lipid nanoparticles’ (LNPs’) size on immunogenicity, biodistribution and cellular uptake represents an important step for enabling the rapid development of novel vaccines and gene therapies [1, 2]. Dynamic light scattering has established itself as an optimal analytical method to determine particle size due to its in-situ approach and fast measurements. However, its application to many systems of industrial relevance has been limited due to artifacts arising from multiple light scattering [3]. In this context, strong sample dilution is often required, bringing additional uncertainties to the formulation development process [4]. Here, we explore how advanced dynamic light scattering technology can filter out multiple scattering and yield fully accurate LNP sizing measurements, regardless of the sample concentration. We seek to illustrate this in a comparative study versus standard dynamic light scattering using polystyrene beads as model suspension as well as a concentrated commercial lipid nanoparticle adjuvant (AddaVax™).

Methods: Measurements in the Modulated 3D cross-correlation scheme (“Modulated 3D DLS”) were performed using a NanoLab 3D (LS Instruments AG). To compare the results with standard backscattering DLS technology, a Malvern ZetaSizer Nano ZS was used as a benchmarking tool (“Standard Backscattering DLS”). Suspensions of polystyrene beads in water were obtained from Bangs Laboratories Inc. at a concentration of 10.06% (solids) and a nominal diameter of 132 nm. A dilution series was conducted to create several sample concentrations down to 0.001%. A squalene-based oil-in-water nanoemulsion of AddaVax™ adjuvant was acquired from InvivoGen with a nominal particle size of around 146 nm and a concentration of 38.7 mg/mL. Dilutions down to 1% volume fraction were prepared using phosphate-buffered saline (PBS; Sigma Aldrich).

Results: Under highly dilute conditions, Modulated 3D DLS and Standard Backscattering DLS both yield an approximately 142nm hydrodynamic diameter. At concentrations above 0.01%, the results differ, with increasing differences at higher concentration. Sizing results with Standard Backscattering DLS indicate measurement error of over 60% at 10% concentration. The observed size reduction is a typical artifact observed with the presence of multiple light scattering events and is not ascribed to an actual particle size change. The Modulated 3D DLS results support this hypothesis; measured hydrodynamic diameter differs by no more than 4% regardless of concentration. Next, a comparison between Standard Backscattering DLS and Modulated 3D DLS was made on the commercial LNP AddaVax™ system. The results measured with Modulated 3D DLS are constant within 2% across all concentrations evaluated. This indicates that the AddaVax™ suspension is stable against dilution. In contrast, the results obtained with the Standard Backscattering DLS technique show a sharp decrease in the measured size with increasing LNP volume fraction, with a maximum measured error of 47%.

Conclusion: The COVID-19 pandemic has brought mRNA technology to the spotlight, and in particular, the use of lipid nanoparticles (LNPs) as a delivery system for mRNA vaccines as well as adjuvants and gene therapies. As particle size is one of the key biophysical parameters for vaccine and therapeutic efficacy and an important indicator of the quality of biologics, it is crucial to be able to accurately determine the sizes, at best within the original formulation. In this work, we have shown that standard Backscattering DLS technology is unable to accurately characterize such formulations. Proper multiple scattering filtering, as performed by Modulated 3D DLS, is required for accurate and reliable measurements in such highly concentrated systems. We have investigated and confirmed this by measurements of a polystyrene bead model suspension and a technologically relevant emulsion system (AddaVax™) formulated at high concentration. The need to use Modulated 3D DLS to suppress artifacts in high concentration systems translates to high-concentration protein formulations and can also provide value for in-vial stability studies and quality control.

References: [1] X. Hou, T. Zaks, R. Langer, and Y. Dong, “Lipid nanoparticles for mRNA delivery,” Nature Reviews Materials, vol. 6, no. 12, pp. 1078–1094, 2021.
[2] A. M. Reichmuth, M. A. Oberli, A. Jeklenec, R. Langer, and D. Blankschtein, “mRNA vaccine delivery using lipid nanoparticles,” Therapeutic Delivery, vol. 7, no. 5, pp. 319–334, 2016.
[3] R. Ragheb, and U. Nobbmann, “Multiple scattering effects on intercept, size, polydispersity index, and intensity for parallel (VV) and perpendicular (VH) polarization detection in photon correlation spectroscopy,” Scientific Reports, vol. 10, no. 21768, pp. 1–9, 2020.
[4] C. Urban, and P. Schurtenberger, “Application of a new light scattering technique to avoid the influence of dilution in light scattering experiments with milk,” Physical Chemistry Chemical Physics, vol. 1, no. 17, pp. 3911–3915, 1999.

Acknowledgements: C. Bretz, A. Vaccaro and D. Leumann were employees of LS Instruments (Switzerland) at the time of the study. A. Jauslin and M. Koch declare no conflict of interest. The work was funded by both companies. A. Jauslin and M. Koch would like to thank the Solvias AG for the support in carrying out the study.

(Top) Particle diameter measured in a polystyrene suspension using Standard Backscattering DLS and Modulated 3D DLS as a function of the volume fraction. (Bottom) Percentage error on the DLS sizing results in both measurement modes, as a function of the volume fraction.

Photograph of AddaVax™ in a measurement cuvette at original formulation concentration (40% volume fraction).

Percentage error of particle size measured in AddaVax™ using Standard Backscattering DLS and Modulated 3D DLS, as a function of the volume fraction.