(T1530-01-04) Multi-Attribute Quantification of mRNA-Lipid Nanoparticle Therapeutics Using Light Scattering

Purpose: We present light scattering techniques that enable high-resolution multi-attribute quantification (MAQ) of free mRNA and lipid nanoparticle-formulated mRNA (LNP-mRNA) in development, manufacturing, and quality control of safe and effective LNP-mRNA therapeutics.

Methods: Multi-angle light scattering (MALS) coupled to field-flow fractionation (FFF) or size-exclusion chromatography (SEC), and dynamic and electrophoretic light scattering (DLS and ELS, respectively.

Results: We provide multiple examples of how MALS coupled to size-based separation by field-flow fractionation FFF or SEC delivers high-resolution quantification of nucleic acid and LNP attributes including size distribution, polydispersity, physical stability, LNP concentration, morphology, and mRNA and lipid concentration. FFF/SEC-MALS not only measures the average quality attributes of an LNP-mRNA sample but can also be used to quantify LNP subpopulations and determine how the mRNA drug substance is distributed across the different particles in the sample. If desired, these subpopulations may be recovered by fraction collection. We also present how FFF-MALS can be used to quantitatively evaluate how different LNP production methods impact LNP properties and potency, and that polydispersity from FFF-MALS can be a more accurate performance predictor for biological models than e.g., hydrodynamic radius or polydispersity from batch DLS. In addition to these high-resolution results, we also discuss how dynamic and electrophoretic light scattering (DLS and ELS) can be used for rapid, low-resolution screening of particle size, concentration, polydispersity, zeta potential and stability as a function of ionic strength, pH, co-solute composition, and other sample properties.

Conclusion: FFF/SEC-MALS provides a 21 CFR Part 11 compliant, automatable solution for LNP multi-attribute quantification that has been validated against conventional offline measurements and that goes beyond the capabilities of laborious low-resolution methods like RiboGreen assays and particle tracking analysis. High-resolution multi-attribute quantification is critical to understanding LNP-mRNA structure-activity relationships and may be used to rationally optimize LNP-mRNA formulation, storage, dosing, and efficacy.