Analyzing the impact of ionizable lipid identity, purity, and stability on lipid nanoparticle performance

Lipid nanoparticles (LNP) have become the leading platform for nucleic acid delivery. However, achieving reproducible and efficacious LNP formulations remains a challenge. Although ionizable lipids are recognized as key determinant for LNP performance, the factors governing efficient RNA delivery are still not fully understood. In this work, we systematically investigated the influence of ionizable lipid identity, purity, and stability on the performance of messenger RNA (mRNA)-loaded LNP. Four structurally different ionizable lipids were evaluated, revealing that transfection efficiency is strongly lipid structure- and cell type-dependent, emphasizing the need of tailoring lipids and, thus, LNP to specific therapeutic applications. Furthermore, batch-to-batch variability and stability studies showed that lipid purity and chemical integrity are critical for LNP's in vitro potency. Lipids containing synthesis- or degradation-derived impurities exhibited markedly reduced mRNA expression in cell culture, despite no apparent differences in "conventional" physicochemical characteristics such as particle size distribution or encapsulation efficiency. Advanced analytical methods, including high performance liquid chromatography with a charged aerosol detector (HPLC-CAD) and liquid chromatography-high resolution mass spectrometry (LC-HRMS), enabled precise detection and quantification of impurities, directly correlating their presence with loss in transfection potency. Overall, this work provides valuable insights into the quality-performance relationships of ionizable lipids utilized in LNP formulations. It underscores the importance of rigorous raw material characterization and stability testing to ensure the reliability of LNP delivery technology.