The efficient purification of extracellular vesicles (EVs) from conditioned cell culture supernatant is critical for their therapeutic application. Various scalable purification technologies are employed, including size exclusion chromatography (SEC), ion exchange chromatography (IEX), and affinity chromatography. Among these, SEC has emerged as a popular and widely adopted method for EV isolation due to its gentle nature, which ensures product integrity.
However, SEC purifies all particles above a specific size threshold. Conditioned cell culture supernatant contains a heterogeneous mix of components, including cell debris, protein aggregates, and other particles that are significantly larger than EVs. These larger contaminants can co-elute with EVs or foul chromatography columns, compromising purity and yield. Therefore, initial clarification and filtration steps are indispensable in the EV manufacturing process. These upstream unit operations effectively remove bulk cellular material and larger debris, ensuring a cleaner starting material for downstream purification techniques like tangential flow filtration (TFF) and SEC and ultimately improving the overall efficiency and purity of the downstream process.
At VivaZome Therapeutics, we have conducted filtration studies to identify suitable filter configurations and pore sizes that efficiently remove contaminants without significant EV loss. Depth filters with multiple layers of progressively finer layers and final retention ratings of 2 to 6 µm were tested for initial clarification of the starting material. Furthermore, we assessed a novel commercial reagent, which prevents filter fouling and significantly increases EV particle recovery, thereby enhancing the scalability and yield.