As a therapeutic tool, extracellular vesicles (EVs) show great promise, both as drug vectors and as treatments in and of themselves. For this promise to be realised, though, the hurdle of producing and isolating at scale must be overcome.
With some studies proposing that more than 1014 EVs could be required for a single therapeutic dose1, the race is on to go from the humble cell culture flask to factory-scale EV production. With yield such a crucial factor, the method of EV isolation need not only be easily scalable but must also enable sufficient EV recovery. In their 2023 paper, Costa et al.2 looked at both of these crucial steps (i.e., culture and isolation), aiming to optimise the production of mesenchymal stem cell EVs for therapeutics. In this article, we will take a look at what they found and discuss how you can make therapeutic-scale EV production and isolation a reality.
Optimising the production of extracellular vesicles from culture media
When it came to production, Costa et al. compared two major factors: static vs stirred-tank bioreactors (STBs), and daily glucose supplementation vs progressive glucose depletion which was tested only in the STBs.
Traditional static culture resulted in the lowest EV production per cell, with just 2.25 × 109 particles per 106 cells, across 7 days of culture. Or, more simply, 2,250 EVs per cell. As half of the media was exchanged on day 4, it is a little hard to identify daily EV production per cell, as this rate will likely have changed over time.
STB culture with steadily declining glucose produced 6,650 EVs per cell, whilst daily glucose supplementation reduced this to only 4,980 EVs per cell. This difference in yield comes despite very similar particle sizes and levels of CD9, CD63 and CD81 between the different culture conditions. For yield then, the use of stirred-tank bioreactors with steadily declining glucose takes the win.
Next the authors added EVs from traditional static culture or STB culture to human umbilical endothelial cells to test the ability of EVs to promote migration and angiogenesis. Of these, EVs from STB culture had the largest impact on promoting wound healing (scratch assay) and tube formation (branching assays), suggesting that pro-angiogenic functionality is improved by using STB culture. There was no comparison to pulsed glucose supplementation for this assay but, given the increase in yield, STB with depleting glucose was chosen as the culture method of choice.
Next it was time to optimise EV isolation.
Tangential flow filtration and size exclusion chromatography vs ultracentrifugation and density gradients
When it comes to isolation, Costa et al. compared their old faithful method of classic ultracentrifugation coupled with density gradient ultracentrifugation (they called this collective method DG-UC) with tangential flow filtration coupled with size exclusion chromatography (TFF-SEC). The SEC in question was a qEV column in the 70 nm series of resin. Figure 1 shows all the detail of how they did this, including the huge time difference between the two techniques. With around 3 hours for TFF-SEC vs 23 hours for DG-UC, the difference in the duration protocol alone could not be starker.
When scaling production, the duration of your EV isolation protocol matters as it impacts upon the possible yield of EVs within a given timeframe. In saying that, having a quick protocol is of no real benefit if EV yield and quality are sacrificed. In this study, EV size was the only EV quality measure applied, and both methods isolated similarly sized EVs. While isolate purity was not specifically addressed, the authors highlighted that there were very low levels of protein in TFF-SEC isolates.
What was addressed, however, was the crucial issue of yield. EV concentration was 5.2-fold higher with TFF-SEC than with DG-UC, making TFF-SEC a more efficient method for scalable isolation of EVs from large volumes of conditioned media – by a significant margin.
Scaling your extracellular vesicle isolation for the clinic
The study by Costa et al. showed that the optimal method for isolating EVs from large volumes is a pairing between TFF and SEC using qEV columns. A recent study in the realm of EVs for diagnostics highlighted that using qEV columns over self-packed SEC columns at scale decreased “technical noise”.3 This is a crucial consideration for therapeutics where batch-to-batch consistency is key. Our qEV PurePath for Therapeutics and Bioprocessing services provide this reduced “technical noise” for the therapeutics space utilising TFF and qEV columns, alongside other techniques. So, what are these services? And how can they help you scale your EV isolation for the clinic?
Our Bioprocessing service uses our facilities to take a large volume of conditioned media or other EV-containing liquid and process it to a pure, high yield, concentrated EV isolate. This will be done using our in-house clarification (e.g., TFF) and the customised (not available off the shelf) large qEV columns paired with our new Zenco chromatography instruments. This is the kind of bioprocessing that startups and academic research laboratories are unlikely to be able to do themselves for a pilot study. The aim of this service is to give you the samples you need to get the data required to take the next steps – be that for further funding, internal approval to scale up or to conduct larger studies testing your therapeutic.
The qEV PurePath for Therapeutics service on the other hand is more of a partnership. With this we aim to partner with you long term to help you from the beginning of your journey all the way through to going into production of a commercial product which can be used to treat patients. With this, you will gain access to our expert scientists and engineers, and we can conduct pilot studies either at your facilities or ours. The instrumentation and workflows perfected in these pilot studies can then be transferred to you to allow you to go into production. Or, alternatively, we can handle production for you. Ultimately, qEV PurePath partnerships are customisable so that you can get your product to patients using a strategy most suited to your unique situation.
Fast-track development by outsourcing EV isolation
Navigating the scale-up process alone can be tough, particularly if the required specialised technology is unfamiliar or inaccessible. Fortunately, we have a wealth of expertise and equipment at our fingertips, meaning you can lean on our bioprocessing team to fast-track your EV isolation scale-up towards a successful clinical product. If this sounds like something that you would be interested in, have a chat with our specialists to get started.
Learn more about outsourcing your EV isolation scale-up
