(W1030-09-57) Development, Qualification, and Application of an LC-CAD-MS Method for Determination of Lipid Content in an RNA-LNP Drug Product Containing Novel Lipids with Diverse Physiochemical Properties

Purpose: The recent success of RNA-LNP therapeutics (Comirnaty, Spikevax, Onpattro, etc.) has led to heightened interest in this therapeutic modality. Interestingly, the lipids in these approved LNPs occupy a relatively limited chemical space. Our organization has developed a lipid library with significant structural variation, and we have demonstrated that including these lipids provides novel functional outcomes such as enrichment in non-hepatic tissues. Though expanding the types of lipids used in RNA-LNPs can provide a variety of exciting functional improvements in delivery, it also creates challenges in the analysis of these more complex drug products. In this poster, we discuss how we developed, qualified, and applied a single LC-CAD-MS assay to assess lipid content and stability in a preclinical RNA-LNP drug product (“NBF-008”) containing a wide range of lipid structures.

Methods: We measure lipid content in RNA-LNP drug product suspensions by disruption/dilution and subsequent analysis by reverse phase LC-CAD-MS. In NBF-008, where the physiochemical space of the lipids used is large, achieving this in a single LC run is challenging. To be able to quantify all lipids and related impurities in a single LC run, we needed to 1) optimize diluent and autosampler temperature to ensure solubility and stability of all components, and 2) optimize mobile phase composition to balance speed, sensitivity, resolution, and pressure. After method development, we qualified the method to demonstrate it was sufficient for IND-enabling studies. We then applied it to temperature-stressed samples to identify key degradants and degradation pathways.

Results: We determined that RNA-LNP dilution in 90% isopropanol, followed by storage in a dark autosampler chamber held at 20°C allowed for continuous solubility and signal stability for anticipated analyte concentrations for at least 48 hr. Elution of all analytes on a single isocrat was not feasible but use of two isocrats with different isopropanol content (56% for smaller/more polar lipids and 80% for larger/less polar lipids) provided sufficient speed (40 min), sensitivity (≤ 0.25% nominal concentration), resolution (R ≥ 1.5 for critical pair), and can be performed at a reasonable pressure (≤ 6000 PSI, column limit). Accuracy, precision, specificity, linearity, and range of the method were confirmed to be sufficient for IND-enabling studies. Finally, we showed that the method could be used to help identify key lipid degradation pathways during storage, in particular hydrolysis, transesterification, and oxidation.br>

Conclusion: Herein, we discuss development, qualification, and application of a single LC-CAD-MS method for determining lipid content in an RNA-LNP (NBF-008) containing lipids with diverse physiochemical properties. We anticipate using this method to quantify lipids and related impurities in the NBF-008 RNA-LNP as this drug product approaches IND submission and Phase I clinical trials. This work provides an example of how well-designed analytical methods play critical roles in the development of novel delivery systems for drug products.