Barnes, Benjamin; (2023) Process Development for Recovery and Purification of Extracellular Vesicles for Biological Products. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Extracellular vesicles (EVs) have rapidly attracted research and commercial interest during the past decade. Potential applications of EVs are diverse and include drug delivery, biomarkers and therapeutics. Despite this growing interest, manufacturing processes for EV production are still in the development phase. The recovery and purification of EVs remain a major hurdle in the progression of fundamental research and the commercial application of EV-based products. Many methods have been described to isolate and purify EVs, each having distinct advantages and challenges. Ultracentrifugation has historically been the most frequently cited EV isolation method, however it is highly operator-dependent, time-consuming and challenging to scale. To this end, the work presented in this thesis explores a scalable and reproducible form of pseudo-affinity chromatography which uses the ligand heparin. Initially, an in-house EV production process was established based on protocols transferred from the project sponsor, ReNeuron. This multi-step process was used to produce concentrated and pre-purified EV material derived from cultures of adherent CTX0E03 neural stem cells or suspension human embryonic kidney cells. This EV material was used as the chromatography column load material in the following three chapters. Initially, heparin affinity chromatography (HAC) using a linear elution gradient was developed for EV purification. This work provides evidence to support an affinity between EVs and heparin, which is currently limited to two conflicting literature articles. This protocol resulted in an EV recovery of at least 68.7%, based on particle counts, which was a 72.6% increase over that using a standard size-exclusion chromatography set-up. Subsequently, a HAC protocol using a step elution gradient was developed for EV fractionation. EV fractionation is a relatively novel development in the EV field that aims to separate EV subpopulations with potentially more desirable properties. This protocol fractionated EVs into two discrete eluted populations that exhibited significantly different tetraspanin immunophenotypes, variable expression of the exosome-associated protein TSG101 and differences in purity. 98.8% of residual protein and 99.0% of DNA were removed using this protocol. Finally, custom chromatography resins were created through the coupling of heparin and related sugars to agarose to gain insights into the structural features of heparin that may be important for EV binding. Specifically, the use of the heparin analogue, tinzaparin, which has a shorter and more consistent chain length, resulted in improved EV binding over the use of standard unfractionated heparin. Overall, HAC shows promise as a method to capture and purify EVs and the work presented in this thesis advances understanding in several important areas from providing evidence to support EV-heparin binding and gaining insights into structural features that facilitate this interaction, to proposing two applications for HAC, EV purification and EV fractionation.

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