Implementation of design of experiments in liposome manufacturing: ethanol injection and extrusion as a case study

Abstract

Although substantial advancements in microfluidics and continuous manufacturing have been made, particularly for lipid nanoparticles (LNPs) used in nucleic acid delivery, conventional liposomes for small-molecule drugs pose distinct challenges. These formulations are typically manufactured via ethanol injection followed by extrusion, a two-step process unlike the integrated nature of microfluidics. This separation introduces additional complexity, as individual unit operations may influence each other, making early-stage optimization more difficult and often requiring iterative refinement across the process. Scaling up these conventional methods remains a challenge, especially in achieving reproducibility, scalability, and regulatory compliance.

Design of Experiments (DoE) provides a valuable framework for understanding and optimizing such processes. However, in the early development stages, particularly in industry, its use is often limited by high material costs, time-consuming analyses, and the need for timely decision-making. Moreover, the existing literature rarely offers detailed, practical guidance tailored to formulation scientists working under these constraints.

This study presents an objective, step-by-step application of DoE to early-phase liposomal formulation development at the 100 mL scale. The goal is not to build highly predictive models, but to rapidly identify robust, workable conditions that enable progression to later development stages. This pragmatic approach reflects the interdependence of manufacturing steps, where downstream processes, such as tangential flow filtration and remote drug loading, may influence or negate prior optimizations.

By evaluating key parameters like ethanol content, stirring speed, and extrusion conditions, the study offers a practical roadmap for resource-conscious process development, supporting informed decision-making in real-world industrial settings.