How do we assess batch-to-batch consistency between extracellular vesicle products?

Jennifer A. Korchak, Elani F. Wiest, Abba C. Zubair

Research output: Contribution to journalArticlepeer-review


Background: Extracellular vesicles (EVs) have gained interest in the field of regenerative and transfusion medicine. Specifically, current Good Manufacturing Practice (cGMP)-grade EVs produced by mesenchymal stromal cells (MSCs) are an intriguing option for cell-free therapeutics. With the development of cGMP-grade EV products, a simple and reliable method for batch-to-batch consistency is needed. Study Design and Methods: The objective of this study was to validate a method to semiquantitatively assess the batch-to-batch consistency of isolated EVs. A multiplex bead-based flow cytometric assay containing 37 surface markers and 2 assay control antibody-coated capture beads was validated. Detection limits (n = 10 buffer samples), repeatability (n = 9 EV samples), and intra-observer reproducibility over 2 days (n = 10 EV batches) were assessed. A Spearman correlation matrix was used to evaluate the batch-to-batch consistency of independently isolated EV products (n = 37 surface markers). Batches with a Spearman correlation coefficient ≥0.9 and p < 0.05 were considered statistically indistinguishable from previous batches. Results: This assay demonstrated robust repeatability as well as intra- and inter-assay reproducibility. In-house batches of EVs were significantly correlated (r ≥ 0.90; p ≤ 1×10−14). Compared with buffer, EV batches had correlation coefficients near zero (r ≤ −0.10; p ≥ 0.12). Commercially sourced EVs significantly correlated with in-house EV batches, but fell below the 90% correlation cutoff (r ≤ 0.71; p ≤ 0.0004). Discussion: This time-efficient and technically simple assay offers a robust method of quality control for assessing the batch-to-batch reproducibility of cGMP-grade EV products.

Original languageEnglish (US)
Pages (from-to)279-287
Number of pages9
Issue number2
StatePublished - Feb 2023


  • clinical translation
  • extracellular vesicles
  • quality control

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Hematology


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