Quality-by-Design (QbD) optimization of lyophilization parameters for siRNA-loaded LNPs using sequential Design of
Manon Degey1, Laura Meloni1, Berfin Dede1
1University of Liège, Laboratory of Pharmaceutical Technology and Biopharmacy, CIRM, 15 Avenue Hippocrate, 4000 Liège, Belgium.
International Journal of Pharmaceutics
|June 3, 2026
Summary
Lyophilization enhances the stability of lipid nanoparticles (LNPs) for RNA delivery. This study optimized the freeze-drying process using a Quality-by-Design approach, ensuring LNP integrity and function after reconstitution.
Area of Science:
- Biotechnology
- Pharmaceutical Sciences
- Materials Science
Background:
- Lipid nanoparticles (LNPs) are crucial for RNA delivery but exhibit poor stability in liquid form.
- Lyophilization (freeze-drying) offers a solution to improve LNP stability during storage.
- Optimizing lyophilization requires understanding cryoprotectant effects and process parameters for LNP integrity.
Purpose of the Study:
- To optimize the lyophilization of siRNA-loaded LNPs using a Quality-by-Design (QbD) framework.
- To identify key factors influencing LNP integrity post-lyophilization and reconstitution.
- To assess the applicability of optimized lyophilization conditions to different LNP formulations.
Main Methods:
- Employed a Quality-by-Design (QbD) framework with two sequential Design of Experiments (DoE).
- Investigated cryoprotectant choice, concentration, and addition mode.
- Optimized freezing and primary-drying parameters for physicochemical preservation and cake appearance.
Main Results:
- Cryoprotectant concentration and addition mode were key drivers of LNP integrity.
- Optimized lyophilization preserved LNP size (200 nm), low Polydispersity Index (PdI), encapsulation efficiency (EE), and in vitro gene silencing.
- Lyophilized LNPs demonstrated extended stability at 4°C and -30°C compared to liquid formulations.
- Optimized process showed formulation-dependent size changes primarily influenced by the ionizable lipid.
Conclusions:
- A QbD framework successfully optimized LNP lyophilization, linking formulation to process outcomes.
- Lyophilization significantly enhances LNP stability, extending shelf-life for RNA delivery applications.
- The developed QbD approach supports rational scale-up and translation of lyophilization processes for diverse LNP formulations.
Keywords:
((4-hydroxybutyl) azanediyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate) (ALC–0315, ionizable lipid)1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, helper lipid)1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE–PEG2000)1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate (SM–102, ionizable lipid)CSL3 (pH–switchable ionizable lipid)CholesterolCritical quality attributes (CQAs)CryoprotectantDimyristoyl-glycerol-methoxypolyethylene glycol-2000 (DMG–PEG2000)LNPLyophilizationQuality-by-Design (QbD)Small interfering RNA (siRNA)StabilitySucroseTrehaloseMore Related Videos
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