Physicochemical and biological evaluation of a GMP‑grade ionizable lipid for robust and scalable manufacturing of siRNA- and mRNA-loaded lipid nanoparticles.

The clinical translation of RNA-loaded lipid nanoparticles (LNPs) requires GMP-grade excipients supported by a rigorous analytical control strategy. This study aimed to evaluate the GMP-grade ionizable lipid (YCE-C10) as a manufacturing-ready component for siRNA- and mRNA-loaded LNPs produced by scalable microfluidic mixing. The evaluated critical quality attributes (CQAs) included Z-average size, polydispersity index, encapsulation efficiency, mRNA integrity, thermotropic behavior, lipid composition, and subvisible particle content. YCE-C10 LNPs exhibited sub-100-nm particle sizes with narrow size distributions, high encapsulation efficiencies, and potent in vitro transfection activity comparable to benchmark formulations prepared with SM-102. Batch-to-batch reproducibility across four independent GMP manufacturing runs was consistent, with relative standard deviations below 5% for all CQAs. LNP encapsulation preserved mRNA integrity across 25-85°C and improved reversibility during cooling compared with naked mRNA. A quantitative HPLC-CAD method demonstrated good linearity for DMG-PEG2K and confirmed consistent lipid composition in formulated LNPs (YCE-C10, 5.21 mg/mL, cholesterol, 4.89 mg/mL, DOPE, 4.13 mg/mL, and DMG-PEG2K, 0.80 mg/mL). Subvisible particles were predominantly <10 µm, with nominal numbers measuring ≥25 µm. In vitro and in vivo studies demonstrated potent mRNA expression and sustained siRNA protection for 24 h, supporting the robustness of the manufacturing platform. The integrated analytical framework of various biophysical methods and in vitro assay is broadly applicable to quality evaluation of LNPs formulated with an ionizable lipid. The findings identify YCE-C10 as a GMP-grade ionizable lipid for reproducible RNA-LNP.