(T1030-02-11) The Importance of Orthogonal Methods for the Determination of the Size Distribution of Lipid Nanoparticles

Purpose: Lipid nanoparticles (LNP) have become the main delivery vehicle for nucleic acids and particularly for mRNA vaccines. Various properties of LNP have been shown to be important for the efficacy of the delivery, including the size of the LNP. The average size and the size distribution of LNP can be modulated by the conditions of their manufacture and it can change during storage or handling. Our goal was to characterize samples with complex size distributions by orthogonal methods to determine the strength and weaknesses of each method.

Methods: We have employed asymmetric flow field-flow fractionation (AF4), dynamic light scattering (DLS), density-gradient centrifugation (DGC), and cryo electron microscopy (cryo-TEM).

Results: Two different sets samples were analyzed: 1) samples where LNP aggregation occurred during freeze/thaw and 2) samples where the LNP were damaged by a surfactant. Each sample set presented different challenges to the employed methods. For the aggregated samples, DLS readily detected the event but could not provide more detailed information. Aggregation was not apparent by cryo-TEM but separation methods like AF4 and DGC showed clearly what has occurred – a small fraction of the LNP aggregated. The surfactant-treated samples presented a different outcome where a significant fraction of the LNP disintegrated after the treatment. The change was the most apparent by cryo-TEM and AF4. DLS-measured size was less able to detect this change, but it was readily detectable as a reduction of DLS signal.

Conclusion: Use of orthogonal methods is critical in particle characterization. Different methods may give different impressions about the sample and only a combination of many methods can provide the full picture.