Abstract 1796: Development of a CSF-1Ri liposomal formulation using a continuous manufacturing approach and design of experiments (DOE)

Abstract Osteosarcoma (OS) predominantly metastasizes to the lungs, with approximately 20% of patients presenting detectable osteosarcoma lung metastases (OSLM) at first diagnosis, while most of the other patients are expected to have undetectable lung micrometastases. OSLM is associated with a 5-year survival rate of only 20%, which not improved over the past four decades. Current OSLM pharmacotherapies are not curative, and experimental immune checkpoint inhibitor therapies have shown minimal efficacy. PLX-3397 (PLX), an FDA-approved colony-stimulating factor-1 receptor inhibitor (CSF-1Ri), targets tumor-associated macrophages (TAMs) in solid tumors, offering a promising strategy to support standard of care in OSLM. However, systemic administration of PLX is limited due to poor tissue distribution and serious off-target toxicities. This study aimed to develop a clinically translatable liposomal PLX (L-PLX) suspension for localized lung delivery via nebulization. A scale-independent continuous manufacturing process and a Design of Experiments (DOE) approach were employed to optimize L-PLX formulations to meet the Quality Target Product Profile (QTPP). Critical quality attributes (CQAs), including hydrodynamic diameter (HD), polydispersity index (PDI), zeta potential (ζ), drug encapsulation efficiency (EE%), and drug loading (DL%), were evaluated. Optimization was performed using A-Optimal DOE with 24 runs, assessing material attributes (MAs) such as Lipid-to-PLX (L:D) ratio and aqueous medium composition, alongside process parameters (PPs) including total flow rate, temperature, and flow rate ratio (FRR).The optimized formulation showed excellent model adequacy with R-squared and adjusted R-squared values of 0.995493 and 0.990575, respectively, confirming its predictive accuracy for particle characteristics. Key factors identified were FRR, L:D ratio, and heating. The optimal formulation, with a L:D ratio of 5, PBS as the aqueous phase, FRR of 5, and room temperature, was validated experimentally, aligning predicted and observed outcomes for particle size, PDI, EE%, and DL%. After additional purification via Tangential Flow Filtration (TFF) to reduce ethanol content to 0.5%, and sterilization, the final product remained stable for over three months at 2-8 ºC, showing consistent HD, PDI, and zeta potential. Aerosol characteristics, tested with a hand-held nebulizer, maintained HD/PDI, suggesting compatibility with pulmonary delivery. Particle size distribution was similar to Arikayce®, the only FDA approved inhaled nanoformulation for lung administration. In vitro experiments with RAW 264.7 macrophages polarized to an M2-like phenotype showed that L-PLX reduced M2-like phenotype, confirming its potential to modulate TAM phenotypes while retaining therapeutic activity. These results highlight L-PLX as a promising candidate for OSLM therapy. Citation Format: Asma M. AlTerawi, Raneem R. Aldaqqa, Fatemah S. Sunbul, David Edwards, Sandro R. da Rocha. Development of a CSF-1Ri liposomal formulation using a continuous manufacturing approach and design of experiments (DOE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1796.