Advanced Quality and Comparability Assessment of mRNA-Loaded Lipid Nanoparticles: Absolute Size Distribution Profiles and Structure from AF4-Coupled Light and X-ray Scattering Measurements

The success of mRNA lipid nanoparticles (LNPs) used in the COVID-19 vaccines has demonstrated the significance of pharmaceutical products utilizing nanoparticle-based drug delivery systems in global healthcare. For the assessment of the safety, efficacy, and quality of these complex, multicomponent systems, it is important to consider not only the properties of the individual components but also their colloidal organization. There is a need for standardized methods to fulfill requirements for application in regular quality control, providing information on these properties in pharmaceutical products. To gain insight into size and size-resolved quality attributes of LNPs, we apply asymmetrical flow field-flow fractionation (AF4) coupled in-line with synchrotron small-angle X-ray scattering (SAXS) measurements, multiangle light scattering (MALS), and UV absorption measurements. We propose model-free algorithms for the analysis of light scattering and X-ray scattering data to obtain quantitative size distribution profiles from both methodologies. The approach is equally applicable for SAXS and MALS data, but SAXS additionally provides detailed, size-resolved insight into internal structure. Information on various quality-indicating parameters for the size-fractionated samples is obtained, including drug loading, internal organization, and particle shape. Since this approach does not require any model assumptions to obtain structural, quality-indicative information from experimental data, it is ideally suitable to evaluate the comparability of results from different systems and different laboratories. This makes it a valuable extension to the regular quality control panel for pharmaceutical nanoparticles, and it should be considered as a standard method in the pharmacopoeias.