Orthogonal pipelines for lipid nanoparticle evaluation

Davidson, Callum and Abdulrahman, Rand and Punnabhum, Panida and Perrie, Yvonne and Rattray, Zahra (2023) Orthogonal pipelines for lipid nanoparticle evaluation. In: SciX 2023, 2023-10-08 - 2023-10-13.

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Ribonucleic acid (RNA) drugs pose promising candidates for gene therapy in treatment resistant conditions and rare diseases. The FDA approval of siRNA Onpattro® in 2018 mRNA-LNP Spikevax® and Comirnaty® COVID-19 vaccinations in 2021[1] ignited research interests as these were the first siRNA and mRNA candidates to utilize lipid nanoparticles (LNPs) as a drug delivery platform. As the RNA-LNP research field is rapidly growing, robust, high-resolution separation techniques coupled to in-line detectors are required to analyze particle key quality attributes and accelerate the successful clinical translation of RNA-LNP therapies. Asymmetric-Flow Field Flow Fraction (AF4) and Size Exclusion Chromatography (SEC) are robust approaches for the characterization of oligo-LNPs [2, 3]. AF4 utilizes perpendicular field induction and particle diffusion-based separation, whereas SEC uses LNP-stationary phase interactions for separation. The goal of this study was to develop separation pipelines for the high-resolution analysis of LNPs. Briefly, we prepared (6Z,9Z,28Z,31Z)-heptatriaconta6,9,28,31-tetraen-19-yl-4-(dimethylamino)- butanoate:cholesterol: 1,2-distearoyl-sn-glycero-3-phosphocholine: 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (MC3:CHOL:DSPC:DMG-PEG2000) LNPs and 8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester:cholesterol: 1,2-distearoyl-sn-glycero-3-phosphocholine: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (SM-102:CHOL:DSPC:DMG-PEG2000) using microfluidics at 50:38.5:10:1.5 mol% ratio. Here, we performed AF4, combined with in-line dynamic light scattering, multi-angle light scattering, and UV detection. Using these detectors, we measured key particle quality attributes including particle size, polydispersity index (PDI), and shape factor. The properties were evaluated alongside oligo-LNP samples that had not been subjected to separation. Manufacture of LNPs using microfluidics-based analysis led to PolyA MC3-LNPs in the 56.5 nm ± 1.2 nm size range with a corresponding PDI of 0.12 ± 0.02, and PolyA SM-102-LNPs of 48.5 nm ± 1.1 nm with a PDI of 0.10 ± 0.01. Our findings show the presence of sub-populations within LNP samples, which cannot be detected using routine particle metrology techniques such as nanoparticle tracking analysis and dynamic light scattering. Our results highlight the need for developing more high-resolution approaches for the analysis of LNPs and linking these to input materials and process parameter design.