Building the Future of Extracellular Vesicle Isolation

As extracellular vesicle (EV)-based diagnostics and therapeutics shift towards and through clinical development, the need for industrial-scale EV isolation workflows becomes increasingly urgent. To learn more about recent progress in this space, we spoke to the CEO at Izon Science, Hans van der Voorn.

Anna MacDonald (AM): Can you tell us about the rationale behind EV-based diagnostics?

Hans van der Voorn (HV): As cell-derived vesicles carrying molecular cargo from the cell of origin, EV offer a fresh approach to biomarker development – mostly as tools for monitoring disease progression and response to treatment. In the case of cancer, tumor-derived EVs have the potential to be monitored as an indicator of how a patient is responding to treatment. Often, people don’t get the right treatment because clinicians don’t have access to real-time data on how treatment is going. However, EVs allow you to look at a relative change, say pre-treatment and after a certain duration of treatment. That is likely to be the biggest use of EVs in the diagnostics space.

Towards this, cargo contained within the bilayer membrane of these vesicles, and molecules attached to the outside of EVs, are being extensively characterized and compared in various healthy and disease states. Profiles of RNA, proteins, metabolites and lipids are all being inspected, with EV-based biomarker development underway for many diseases and medical applications. Through the work of our partners and customers we are seeing progress in biomarker discovery and validation continue for many cancers, maternal-fetal medicine and supporting fundamental biological research. While these are the main areas of research, our diagnostics-focused customers also span agriculture and even conservation.

AM: How can EVs be applied as therapeutics?

HV: Inherently, EVs are attractive vehicles for therapeutic delivery, and they are also being explored in the cosmetics industry. As naturally occurring vesicles they are well tolerated in vivo and also protect cargo from degradation. Work is underway to see how these properties can be leveraged. With EV-based therapeutics, there’s a very wide variety of ways that EVs can be applied, either as drug delivery vehicles, or as therapeutics themselves. EVs can be harvested from cell culture, so there’s work around stem cell-derived EVs, EVs loaded with particular RNAs and surface-modified EVs. Regardless of the approach, they all need the same thing – consistent methods of EV isolation with high efficiency, repeatability and scalability, and you need to be able to analyze what you’re getting out of them. 

AM: What are some of the key challenges facing the development of EV applications?

HV: Although the field has come a long way, EVs are difficult to study in vivo, so verifying their biological functions is difficult. One key theme across the field boils down to "reproducibility" – how can EVs be isolated and analyzed in a way that produces results replicable by others, when there are so many variables? EVs are heterogenous in size and origin and exist in biofluids which are inherently complex, and their composition can be altered by different sample collection and storage protocols, for example.

With that in mind, EV isolation becomes very important, as it's going be very difficult to discover and validate a biomarker, or test a therapeutic proof-of-concept, if you haven't been processing your sample in a highly consistent manner. Reproducible, efficient isolation sets the groundwork for everything else and is critical regardless of whether you're working with a small or large number of samples. The other factor to consider is scalability – the isolation method of choice needs to be scalable, to accommodate a high-throughput of samples in diagnostics, or a large volume of cell culture media for therapeutics.

AM: How is Izon Science addressing the need for reproducible EV isolation?

HV: To date, this has been through the qEV Isolation platform, which consists of size exclusion chromatography-based qEV Columns to separate EVs from soluble protein. qEV Columns can be paired with the Automatic Fraction Collector (AFC), a programmable instrument that automates the collection of your purified EVs. Initially the AFC was invented as a labor-saving device, but it ended up delivering a level of precision and reproducibility that wasn’t there before.

Traditionally, people collected a whole lot of fractions and analyzed the EVs in them separately, but we are proposing that, ultimately, you need to focus on one EV-containing fraction. You might spend a bit of time optimizing exactly which part of the volume that fraction is, but that’s the way forward – just collecting one fraction, not multiple fractions.

With customers needing to isolate EVs from a wide range of sample volumes, we grew the column range to accommodate the broad spectrum of EV research. While our standard columns cover sample loading volumes from 150 µL to 100 mL, we recently got a contract for isolating EVs from 200 liters. Considering our smallest columns accommodate sample loading volumes of 150 µL, that’s hugely different from starting volumes of 200 L – a difference of 10^6, and a reflection of diversity in the field.

AM: What exciting projects are you working on?

HV: Our goal is to simplify isolation for the end user. As our customers all have very diverse goals, there’s a whole array of things we are doing. We are working on what we think is the largest EV isolation project in the world, in terms of sample volume, which involves total customization and the optimization of several different processing and isolation technologies. Separate to that is the possible development of an 800 nm filter, which was identified through joint research with our partners at AUMC (Amsterdam University Medical Center). The notion is that there are still cell fragments present in blood samples, so a pre-SEC filter step may help further improve isolation. Research is ongoing and the filter hasn’t found its way into commercial development but I expect it will. We expect that the optimal solution will include an 800 nm filter, followed by SEC, followed by concentration of the purified EV-containing isolate.

It's important to distinguish that we are focused on specific isolation – enabling customers to separate their particles of interest from other components in the sample. That is distinct from specific measurement, which is not what we are trying to achieve. Towards this, we are experimenting with different resins that go into our qEV columns, which will give people the ability to further refine their EVs of interest through specialized isolation columns. In addition, we are advancing the instrumentation that goes with the columns, to enable qEV columns to be primed and run in parallel.

AM: How will approaches to EV isolation change as EVs head towards the clinic?

HV: The isolation requirements for EV-based diagnostics and EV-based therapeutics are very different, so the future of EV isolation will split into two pathways. For this reason, we recently launched two services: qEV PurePath for Diagnostics and qEV PurePath for Therapeutics. Growing companies focused on developing clinical applications of EVs have enough on their plate, and developing scalable and efficient isolation methods is just one of them. Outsourcing EV isolation R&D through services like qEV PurePath for Therapeutics and qEV PurePath for Diagnostics therefore offers a viable solution. These services aim to streamline and tailor EV isolation workflows, enabling researchers and companies to concentrate on refining their EV-based products.

In diagnostics, high-throughput instrumentation will allow qEV Columns to be primed and utilized in parallel, for the isolation of multiple samples at once. In the therapeutics space, size exclusion chromatography forms one part of a wider bioprocess for processing large cell culture volumes. Those looking for GMP-ready qEV columns have extra requirements, so we’ve also been ramping up QC measures and setting up the capacity for additional microbial testing. In summary, future technology will reflect increasingly divergent requirements and a need for expert customization.

Hans van der Voorn was speaking to Anna MacDonald, Senior Science Editor for Technology Networks.