A Scalable Continuous Production Platform for Large-Scale Manufacturing of Therapeutic Exosomes

Project Summary Exosomes are a subset of extracellular vesicles, with diameters between 30 and 200 nm, secreted by most eukaryotic cells. They deliver bioactive cargos, such as lipids, proteins and nucleic acids, to other target cells. Tremendous effort has been made recently on developing highly specific exosome-based

drug delivery systems. Yet, there remain fundamental challenges: 1) current technologies for exosome enrichment and isolation cannot achieve sufficient throughput or purity; 2) drug loading efficiency into exosomes is very limited; and 3) the production of exosomes has yet to reach sufficient high throughput

for clinical tests or even further development. To address these challenges, the proposed project utilizes a three-dimensional (3D) piezoelectric scaffold (PES) to stimulate exosome production, a lipid- membrane penetrating chiral graphene quantum dots (GQD) for efficiently load drugs into exosomes (>70% yield) and a scalable tangential-flow ultrafiltration module with ion-track asymmetric nanopore

membranes (ANM) to isolate exosomes with high yield (>85%), throughput (240 mL/hour per module) and purity. The integration of three modules constitutes a high-throughput, high-purity, and scalable integrated platform for continuous manufacturing of therapeutic exosomes. The project is based on PI Wang’s preliminary experimental and computational results that GQDs facilitated drug loading into

exosomes with higher efficiency than any current method, her extensive experience on nanocomposite for 3D tissue cultures, the co-PI Chang’s ANM exosome isolation technology for liquid biopsy and his patented technology on high-throughput AC nanofiber electrospinning. We will achieve the goal by following specific aims: 1) Develop high-throughput exosome production bioreactor module with

stimulating PES. 2) Develop high-efficiency exosome drug loading module with chiral GQDs. 3) Develop and establish continuous ANM isolation and purification module. The proposed project contains several innovated approaches of exosome production, loading and isolation, if successful and integrated, can enhance therapeutic exosome production rate over current design, and achieve

continuous production of therapeutic exosomes. Such an integrated production pipeline provides an innovative high-throughput and high-purity exosome manufacturing platform for drug delivery. They will also be designed to allow easy scale up for reactors of arbitrary size or for effluents from multiple

reactors. Scaling to higher throughput can be achieved by just adding more parallel lines of the same modules.