Application of quality by design to formulation and processing of protein liposomes.

Quality by design (QbD) principles were explored in the current study to gain a comprehensive understanding of the preparation of superoxide dismutase (SOD) containing liposome formulations prepared using freeze-and-thaw unilamellar vesicles (FAT-ULV). Risk analysis and D-optimal statistical design were performed. Of all the variables investigated, lipid concentration, cholesterol mol%, main lipid type and protein concentration were identified as critical parameters affecting SOD encapsulation efficiency, while the main lipid type was the only factor influencing liposome particle size. Using a model generated by the D-optimal design, a series of three-dimensional response spaces for SOD liposome encapsulation efficiency were established. The maximum values observed in the response surfaces indirectly confirmed the existence of a specific SOD-lipid interaction, which took place in the lipid bilayer under the following optimal conditions: (1) appropriate membrane thickness and curvature (DPPC liposomes); and (2) optimal "pocket size" generated by cholesterol content. With respect to storage stability, the prepared SOD liposomes remained stable for at least 6 months in aqueous dispersion state at 4°C. This research highlights the level of understanding that can be accomplished through a well-designed study based on the philosophy of QbD.