Extracellular vesicles (EVs) from stem-like cells are emerging as therapeutic modalities due to their regenerative and anti-inflammatory properties. For clinical translation of EVs, standardisation of EV manufacturing and analytical assays is required. This is guided by the development of a Quality Target Product Profile (QTPP), underpinned by the identification of Critical Quality Attributes (CQAs) such as physical, chemical, and biological characteristics of purified EVs. Defining acceptable ranges and distribution of these CQAs ensures each batch meets the requirements of the QTPP.
This study focuses on developing and standardising assays to quantify and characterise VivaZome’s proprietary cell-derived EVs (VZT-PC-EVs) and establish their CQAs. Each batch is assessed for i) particle concentration, size distribution, and zeta potential (ZetaView Nanoparticle Tracking Analysis (NTA)), ii) surface marker expression (fluorescence-NTA), iii) total protein, RNA, and DNA (Qubit Fluorometry), and iv) other key markers (Western immunoblot). From four consecutive batches, protein removal was >99%, with a particle-to-protein ratio >5E+08, indicating high purity. Additionally, the particle-to-DNA and particle-to-RNA measures were >1E+12 and >1E+11 respectively. The median size had a coefficient of variance of 6%, confirming batch consistency. Zeta potential measurements were also consistent across batches. Further characterisation of VZT-PC-EVs has been performed using cryo-TEM, proteomics, and RNA sequencing, revealing additional markers, such as microRNAs, and attributes that contribute to defining CQAs and ensuring batch-to-batch consistency. By integrating industry-driven development with academic innovation, future work will advance the analytics required for manufacturing and analysing therapeutic EVs.