Bringing cell and gene therapy drugs to market offers a chance for curing previously intractable diseases, but it comes with a formidable challenge of ensuring the safety and efficacy of increasingly complex therapeutics. These drugs are multicomponent ensembles (carrier + payloads), which means there are an array of critical quality attributes (CQAs) that must be monitored at each stage of their development – creating bottlenecks and delaying the process. High-throughput analytical methods that permit multiattribute measurements constitute a potential solution to this issue. Among the most efficient are chromatographic separations that can be coupled to advanced detectors, as with size exclusion chromatography paired with multiangle light scattering (SEC-MALS), which is of growing importance to emerging new modalities (AAVs, mRNA/LNPs etc.).

Significant attention is being placed on creating fit-for-purpose SEC methods for lipid nanoparticles (LNPs) and their payload drug substances. Optimized widepore, low adsorption SEC columns (>450 Å) have been developed such that new techniques can be envisaged. Past investigators have applied a positive surface potential gel permeation resin to perform LNP size exclusion chromatography. There is risk to this approach due to the potential adsorption and incomplete elution of free nucleic acid impurities. As such, we have alternatively investigated non-ionic, hydrophilic packing materials for SEC of LNPs. We have compared the recovery of the LNP materials across different columns and found optimal conditions that led to over >75% recovery with minimal tailing allowing for MALS mediated size, mass and heterogeneity measurements. Additional investigations are underway to further improve our understanding of analyte of recovery.

Moreover, we developed conditions for a complete online disruption of the LNP carrier which enables a direct measurement of the nucleic acid payload(s) with a 2 ng as the limit of detection (LOD). We show that efficient size based separation is crucial for dual payload quantitation of gene editing therapy oriented LNPs containing both mRNA and gRNA. Various denaturing agents and LNP formulations (>5) were investigated to ensure universality of the disrupting conditions and the creation of a platform method.

In summary, we demonstrate the versatile use of SEC-MALS to extract multiple CQAs (purity related e.g. RNA integrity or presence of process related impurities or strength related such as mRNA content) measurements for LNP RNA drug products and drug substances. With its deployability, SEC-MALS is coming to establish a cost and time-effective means to comprehensively characterizing a range of new nucleic acid therapies.