The development of extracellular-vesicle-based therapeutics requires robust analytical assays and scalable downstream processing strategies to ensure product quality and reproducibility. In this study, we established an analytical toolbox comprising scattering and fluorescence mode nanoparticle tracking analysis, fluorescence-based flow cytometry, and multidetector analytical size-exclusion chromatography. Liposomes, selected for their biophysical similarity to EVs, were used as reference materials to optimize assay parameters, fluorescence labeling conditions, and dynamic range, minimizing artifacts such as photobleaching and masking effects. Using these optimized analytical tools, we established a scalable downstream processing (DSP) workflow for human bone marrow mesenchymal stromal cell-derived EVs (MSC-EVs), incorporating clarification, tangential flow filtration, ion exchange chromatography, buffer exchange, and sterile filtration. We first designed and optimized the DSP process at small scale using ion exchange chromatography (IEX). We integrated controls such as medium-alone control to ensure the cell-secreted nature of the final preparation. Using a stepwise elution strategy, IEX chromatography resulted in the elution of two cell-secreted populations. We then investigated the identities of these two IEX eluates using orthogonal analytical techniques. Transmission electron microscopy revealed that eluate 2 was enriched in EVs with a cup-shaped morphology, while eluate 1 contained nonvesicular extracellular particles (NVEPs). Eluate 2 also showed higher expression of positive markers such as CD81 and CD73 using Simple Western and expressed MSC-specific RNA markers compared with eluate 1. Proteomic and lipidomic analyses further validated the enrichment of EVs in eluate 2. Then, the analytical toolbox was utilized to monitor the DSP process for particle recovery and impurity removal throughout the process. Together, these results demonstrate that orthogonal analytics coupled to a scalable DSP yield reproducible MSC-EV preparations while depleting commonly co-isolated NVEPs. This practical framework advances process analytics of MSC-EV manufacturing and supports reporting of identity, purity, and function, aligned with existing guidelines in the field.
Dehghani et al. (Mon,) studied this question.