Design and Application of Affinity-Based Tools for the Selective Capture of Native and Engineered Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are increasingly recognized as valuable tools for diagnostics and therapeutic delivery, yet scalable isolation and consistent purity remain major challenges. Contaminants co-isolated with EVs can compromise their biological function and therapeutic reproducibility. This thesis explores affinity-based strategies for improving the selectivity, reproducibility, and scalability of EV purification.Two complementary approaches were investigated. The first involved the use of recombinant affinity ligands recognizing abundant EV surface markers to capture native vesicles directly from cell culture supernatants. These ligands were expressed in E. coli, purified, and applied for EV isolation from suspension-adapted human cell lines. Comparative analyses confirmed enrichment of canonical EV markers and high purity based on particle-to-protein ratios, supporting the suitability of this approach for standardized EV preparations.The second approach focused on engineering EV-producing cells to enable selective capture and gentle release of vesicles through affinity interactions. Constructs were designed to allow controlled removal of purification elements following isolation, preserving vesicle integrity and functionality. The resulting engineered EVs exhibited monodisperse size distributions and robust recovery across multiple process conditions.In both workflows, alternative affinity resins and elution strategies were benchmarked to identify combinations supporting efficient purification and reusability of chromatographic material. The optimized process achieved high yields and purity metrics consistent with current standards in the field.Overall, the study demonstrates two scalable affinity-based strategies that enhance the purity and recovery of extracellular vesicles, providing a basis for further development of reproducible and industry-compatible EV manufacturing processes.