Proof of Concept Application of Hydrophilic Interaction Chromatography for Direct Online Disruption of Lipid...

Lipid nanoparticles (LNPs) are a key platform for delivering mRNA vaccines and therapeutics with numerous innovative drugs under development. However, characterizing these complex and unstable products remains challenging. Developing fast, reliable methods to assess critical quality attributes (CQAs) of the mRNA component is crucial for ensuring the safety and efficacy of these medicines. Currently, evaluating key CQAs, such as mRNA integrity and encapsulation efficiency, often involves a labor-intensive manual extraction protocol, which requires LNP disruption prior to analysis. However, these additional offline steps contribute to mRNA degradation and measurement uncertainties, highlighting the urgent need for rapid and effective methods capable of performing an online LNP disruption. Hydrophilic interaction chromatography (HILIC) might offer a promising solution to address this need. Due to the presence of high concentrations of organic solvent and the possibility to work at elevated temperatures, HILIC might enable on-column disruption of LNPs while preserving the full integrity of the mRNA payload, facilitating a streamlined characterization process. To evaluate this, we developed two proof of concept HILIC methods. The first one disrupts LNPs and retains the mRNA payload using a high percentage of organic solvent and elevated temperatures. The second one, relying on milder conditions, retains only the unencapsulated mRNA, which can be used to evaluate the encapsulation efficiency. Both methods were used on Comirnaty and Spikevax vaccines and on Sanofi's in-development mRNA product as model samples. Our preliminary findings suggest that HILIC holds potential for online LNP disruption, mRNA integrity assessment, and encapsulation efficiency analysis. They also highlight the limitations of small-pore-sized columns currently available on the market.