UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Development of a Process Step for the High Purity Recovery of Exosome Material from a Regenerative Cell Product

Colao, Ivano Luigi; (2021) Development of a Process Step for the High Purity Recovery of Exosome Material from a Regenerative Cell Product. Doctoral thesis (Eng.D), UCL (University College London).

[thumbnail of Development of a Process Step for the High Purity Recovery of Exosome Material from a Regenerative Cell Product_vivacorrections.pdf] Text
Development of a Process Step for the High Purity Recovery of Exosome Material from a Regenerative Cell Product_vivacorrections.pdf - Accepted version
Access restricted to UCL open access staff until 1 January 2022.

Download (7MB)

Abstract

Exosomes are an emerging sub class of extracellular vesicle which are rapidly gaining momentum as a novel therapeutic platform. Their regenerative and therapeutic potential is reflective of the plethora of cell types from which they can be derived. However, as the technology is in its nascent stage, relatively little exists in terms of defined manufacturing processes. As exosome technologies progress towards pre-clinical and clinical stages, the constraints in manufacturing processes, specifically in downstream processing, will need to be overcome. The current “gold standard” for exosome recovery from conditioned culture medium is ultracentrifugation. This step is time consuming, prone to operator error and difficult to scale and translate. The work presented here shows that monolith chromatography is a scalable, and reproducible purification option which can be used to successfully recover functionally active, and highly pure exosomes. Exosomes derived from the clinically relevant stem cell line, CTX0E03, were shown to present the biomarkers CD 9, CD 63 and CD 81, commonly conserved amongst exosome species throughout the literature. The vesicles were characterised as having a size distribution between 20 to 150 nm, and a flotation density between 1.136 – 1.185 g mL-1, as expected based on literature values. Furthermore, exosomes recovered by tangential flow filtration (TFF), were shown to promote fibroblast migration and wound closure (98% ± 1.5%) in an in vitro potency model, in a dose dependant fashion. In contrast exosomes purified by ultracentrifugation could not achieve wound closure, with no significant difference observed over the 72 hour period. TFF recovered exosomes were purified by the processes developed in this thesis. In the first instance they were purified by use of an anion exchange monolith using the quaternary amine ligand. Exosomes were shown to elute broadly over the elution gradient and overlap with DNA and albumin co-present within the feed material. Samples obtained post purification had purity ratios of 1.5 x 10^9 particles per µg of protein and 9.3 x 10^11 particles per µg of DNA impurity. Based on a hypothetical dose size of 10^9 particles per mL this result indicated purities within the WHO guidelines for injectable therapeutics (100 µg of protein, 10 ng DNA per dose) and benchmarked a potential method for purification of exosomes. A second monolith was also tested, using an orthogonal chemistry: hydrophobic interaction with an OH ligand. The results of this column surpassed those of the AEX process and showed a binding affinity beyond the hypothesized values. Unlike the AEX column, the HIC operation did not co-bind impurities in the form of albumin, DNA or even cell-0derived organelle matter. Resultantly, purities were even higher than those of the AEX column at 3.97 x 10^9 particles per µg of protein, and 3.12 x 10^12 particles per µg of DNA. Finally, combination of the processes showed the potential application of the chromatographic options within a larger process for exosome purification and high performance capillary electrophoresis analysis showed substantial removal of cell culture derived proteins from the recovered material, without substantial loss in particle number. The processes were assessed for potency, both individually and in sequence. No adverse effect in wound closure was noticed with all samples achieving wound closure over 90%. This showed improvement on the current gold-standard method, which could not retain product functionality.

Type: Thesis (Doctoral)
Qualification: Eng.D
Title: Development of a Process Step for the High Purity Recovery of Exosome Material from a Regenerative Cell Product
Event: UCL
Language: English
Additional information: Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
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
URI: https://discovery.ucl.ac.uk/id/eprint/10129235
Downloads since deposit
3Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item