Although lipid nanoparticles (LNPs) are widely used for RNA delivery, their use is limited by poor stability in liquid form during refrigerated storage. Lyophilization is a promising way to address this limitation. However, the relationship between cryoprotectant choice and process settings in determining LNP integrity after reconstitution is not yet fully understood. It also remains unclear whether conditions optimized for one formulation can be applied to others. Here, a Quality-by-Design (QbD) framework was used to optimize the lyophilization of siRNA-loaded LNPs using two sequential Design of Experiments (DoE). The main drivers of post-lyophilization LNP integrity were found to be cryoprotectant concentration and mode of addition, while freezing and primary-drying durations required fine tuning to balance physicochemical preservation and cake appearance. Under the selected condition, lyophilized CSL3/DSPE-PEGLNPs showed, after reconstitution, a Z-average size of 200 nm with a remaining low Polydispersity Index (PdI), maintained siRNA encapsulation efficiency (EE), preserved nanoscale organization, and maintained in vitro gene silencing efficiency. Compared with significant deviation from initial critical quality attributes (CQAs) within 13 days in the liquid state, lyophilization substantially extended stability at 4 °C and enabled preservation through 11 weeks at -30 °C. Applying the optimized process to LNPs with different lipid compositions showed that lyophilization-related size changes were mainly driven by the ionizable lipid, while the lipid-PEG anchor had only a minor effect. Overall, this study provides a QbD framework that links formulation composition to lyophilization outcomes and supports rational translation of the process across LNP formulations. 2000
