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GFP-tagging of extracellular vesicles for rapid process development

Carrillo Sanchez, Braulio; Hinchliffe, Matthew; Bracewell, Daniel G; (2022) GFP-tagging of extracellular vesicles for rapid process development. Biotechnology Journal , Article e2100583. 10.1002/biot.202100583. (In press). Green open access

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Abstract

Extracellular vesicles (EVs) act as nano-scale molecular messengers owing to their capacity to shuttle functional macromolecular cargo between cells. This intrinsic ability to deliver bioactive cargo has sparked great interest in the use of EVs as novel therapeutic delivery vehicles; investments totaling over $2 billion in 2020 alone were reported for therapeutic EVs. One of the bottlenecks facing the production of EVs is the lack of rapid and high throughput analytics to aid process development. Here CHO cells have been designed and engineered to express GFP-tagged EVs via fusion to CD81. Moreover, this study highlights the importance of parent cell characterization to ensure lack of non-fused GFP for the effective use of this quantitative approach. The fluorescent nature of resulting vesicles allowed for rapid quantification of concentration and yield across the EV purification process. In this manner, the degree of product loss was deduced by mass balance analysis of ultrafiltration processing, reconciled up to 97% of initial feed mass. The use of GFP-tagging allowed for straightforward monitoring of vesicle elution from chromatography separations and detection via western blotting. Collectively, this work illustrates the utility of GFP-tagged EVs as a quantitative and accessible tool for accelerated process development.

Type: Article
Title: GFP-tagging of extracellular vesicles for rapid process development
Location: Germany
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/biot.202100583
Publisher version: https://doi.org/10.1002/biot.202100583
Language: English
Additional information: © 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Keywords: CHO cells, extracellular vesicles, nanobiotechnology, process analytics, process development, size exclusion chromatography, ultrafiltration
UCL classification: UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10146760
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