Large-scale manufacturing of immunosuppressive extracellular vesicles for human clinical trials
Meizhang Li, Rupal P. Soder, Sunil Abhyankar, Trisha Home, Harsh B. Pathak, X M Shen, Andrew K. Godwin, Haitham Abdelhakim
Abstract
BACKGROUND: Human mesenchymal stromal cells (MSCs), particularly Wharton's jelly-derived MSCs (WJMSCs), offer significant therapeutic potential for complex immune conditions such as graft versus host disease (GVHD), in part through their secreted small extracellular vesicles (sEVs). These sEVs exhibit crucial immunomodulatory properties, including suppression of T-cell activation demonstrated both in healthy donor cells and in pathologic contexts. Despite this promise, widespread clinical application is impeded by substantial challenges in developing robust, scalable, and Good Manufacturing Practice (GMP)-compliant manufacturing processes for MSC-derived sEVs to meet clinical demand. METHODS: To address the critical barriers in sEV production, this study details the development and validation of a reliable and scalable manufacturing platform for WJMSC-derived sEVs. The process utilized GMP expanded WJMSC culture medium, generated from cultured low-passage cells and processed in scalable 2-L and 6-L batch volumes. This platform employed a sequential approach involving tangential flow filtration (TFF) for efficient initial concentration followed by size-exclusion chromatography (SEC) for comprehensive final purification of sEVs. RESULTS: The integrated TFF-SEC manufacturing approach resulted in a significant enrichment of nanoparticles, demonstrating up to a 16.9- and 36-fold increase in particle concentration post-TFF from the 2-L and 6-L batches, respectively. Two batches of purified sEVs demonstrated very similar mean size ranges, from 142 ± 2 nm to 156 ± 2 nm, and displayed the markers CD9 and CD81 while not expressing the negative marker calnexin. Those WJMSC-derived sEVs maintained their biological activity, effectively suppressing - cell activation in vitro. Furthermore, the purified sEVs from both 2-L and 6-L batches demonstrated an intact structure observed by cryogenic electron microscopy (cryo-EM), positivity for the inhibitory immune checkpoint ligand PD-L1. CONCLUSION: In this study, we report a reliable large-scale manufacturing framework that combines TFF and SEC to manufacture WJMSC-derived sEVs. The established standard operating procedures (SOPs) will help guide the design and establishment of industrial-scale, clinical-grade WJMSC-derived sEV manufacturing. This work significantly advances the field by offering a practical pathway that is anticipated to facilitate the broader development and accelerate the clinical translation of these WJMSC-derived sEVs as potent, cell-free therapeutic agents for various human diseases.