A Comprehensive Evaluation of Analytical Method Parameters Critical to the Reliable Assessment of Therapeutic mRNA Integrity by Capillary Gel Electrophoresis.

In recent years, messenger ribonucleic acid (mRNA)-lipid nanoparticle (LNP) biotherapeutics have demonstrated significant promise in disease treatment and prevention given their rapidly modifiable production processes and considerable capacity to adapt to complex or low-yielding proteins of interest. As a result, many products are currently being developed in this space. Critically, well-characterized and appropriately designed assays are required to monitor purity and integrity in order to maintain the efficacy and consistency of these novel products. Currently, capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ion-pair reversed-phase liquid chromatography (IP-RPLC) are techniques of choice for mRNA integrity analysis. However, most methods proposed for biotherapeutic analysis have been developed using naked mRNA without LNP components or proprietary buffer formulations, which can obscure undiscovered impurities or complex interactions between mRNA and the sample matrix. In this study, we addressed these methodological challenges by using a biotherapeutically relevant commercial mRNA-LNP sample (approx. 4200 b) to refine and optimize a customizable CGE-LIF method currently under consideration for mRNA-LNP biotherapeutic analysis. We systematically characterized how critical method parameters-such as denaturant type, concentration, and usage-and LNP disruption protocols can interfere with accurate mRNA integrity analysis in CGE-LIF and IP-RPLC. We found that optimal conditions for CGE-LIF assay sensitivity, variability, and resolution included sample precipitation by isopropanol, high urea concentrations, no formamide as a sample diluent, and high concentrations of dye. Finally, the advantages and disadvantages of both CGE-LIF and IP-RPLC are highlighted, and a discussion of key considerations when using or designing methods for mRNA integrity assessment is presented.