Messenger RNA (mRNA)‐based vaccines are advantageous because they can be relatively quicker and more cost efficient to manufacture compared to other traditional vaccine products. Lipid nanoparticles have three common purposes: delivery, self‐adjuvanting properties, and mRNA protection. Faster vaccine development requires an efficient and fast assay to monitor mRNA purity and integrity. Microchip CE is known to be a robust technology that is capable of rapid separation. Here, we describe the development and optimization of a purity and integrity assay for mRNA‐based vaccines encapsulated in lipid nanoparticles using commercial microchip‐based separation. The analytical parameters of the optimized assay were assessed and the method is a stability indicating assay.

The development of mRNA vaccines has increased rapidly since the COVID-19 pandemic. As one of the critical attributes, understanding mRNA lipid nanoparticle (LNP) stability is critical in the vaccine product development. However, the correlation between LNPs’ physiochemical characteristics and their potency still remains unclear. The lack of regulatory guidance on the specifications for mRNA LNPs is also partially due to this underexplored relationship. In this study, we performed a three-month stability study of heat-stressed mRNA LNP samples. The mRNA LNP samples were analyzed for their mRNA degradation, LNP particle sizes, and mRNA encapsulation efficiency. In vitro cell potency was also evaluated and correlated with these above-mentioned physiochemical characterizations. The mRNA degradation–cell potency correlation data showed two distinct regions, indicating a critical cut-off size limit for mRNA degradation. The same temperature dependence was also observed in the LNP size–cell potency correlation.

Background/Objectives: Host cell protein (HCP) content is a major attribute for biological and vaccine products that must be extensively characterized prior to product licensure. Enzyme Linked Immunosorbent Assay (ELISA) and Mass Spectrometry (MS) are conventional methods for quantitative host cell protein analysis in biologic and vaccine products. Both techniques are usually very tedious, labor-intensive, and challenging to transfer to other laboratories. In addition, the ELISA methodology requires 2D SDS PAGE and 2D western blot antibody reagent validation to establish reagent coverage. This reagent coverage provides a rather weak link that is currently accepted, as the western blot is run under denaturing conditions and the ELISA is run under native conditions. Simple Western™ is a relatively new, automated, capillary western blot-based technology that allows for the separation, blotting, and detection of proteins. But, unlike traditional western blots, Simple Western™ is quantitative, allowing for the quantification of HCP content in biologic and vaccine samples. Antibody reagent validation is much more straightforward, as the reagent coverage can be directly linked between the 2D methodology and Simple Western™, as they are both run under denatured and reduced conditions. Methods: Herein we describe the development of a capillary western blot method to quantify the HCP content in samples generated using a Vero cell line for the production of an investigational live virus vaccine candidate (V590) for Coronavirus Disease-2019 (COVID-19). The HCP content in COVID-19 vaccine samples was evaluated using three methods: the new capillary western, the gold standard ELISA, and SDS-PAGE. Results/Conclusions: Strong agreement was observed in the HCP content data between the capillary western and SDS PAGE methods, whereas the ELISA HCP data were outliers, suggesting that the capillary western is generating HCP concentrations closer to the true concentration. This is the first report of using capillary western technology in analyzing HCP in vaccine samples.