Extracellular vesicles (EVs) obtained from liquid biopsies are potential biomarkers for a huge range of diseases, from cancer1 to neurodegenerative disorders2. However, biofluids contain much more than just EVs, including an abundance of protein and lipid-based particles that can contaminate EV isolates. Achieving a high yield of sufficiently pure EVs has proven challenging for applications such as diagnostic test development.
Size exclusion chromatography (SEC) has rapidly become the isolation method of choice in the EV field, particularly for diagnostic research3. Major advantages of SEC include its ability to process clinically relevant (i.e. small!) sample volumes4, its preservation of EV integrity5, and its ease and speed of use6. Unfortunately, lipoprotein contamination of EV-containing SEC fractions has been reported as a problem in standard SEC7 because EVs and lipoproteins, such as chylomicrons and very low-density lipoproteins (VLDLs), fall within the same size range. To address this, Izon developed SEC+, an enhanced SEC approach that delivers purer EV isolates without compromising yield.
On-column lipoprotein removal
SEC+ leverages our exclusive Gen 2 resin formulation to separate particles based on size, whilst also retaining positively charged particles within the column. Since EVs are more negatively charged than lipoproteins8, our Gen 2 SEC+ columns can efficiently separate EVs and lipoproteins, even when the particles are similarly sized. This significantly reduces lipoprotein contamination in EV isolates as compared to standard SEC, such as our now discontinued Sepharose-based Legacy columns (Figure 1). In our in-house studies, the qEV Gen 2 / 70 nm Series removed an average of 99% of ApoB (found in chylomicrons, IDL, LDL and VLDL) while the qEV Gen 2 / 35 nm Series removed 94%. Legacy columns, which separate on size alone, removed an average of 97% and 80% of ApoB using 70 nm and 35 nm columns respectively.
Trade off or win-win?
Improvement in one performance area can often come at the expense of another. So, let’s check in on protein removal and EV yield using Gen 2 SEC+ columns. Gen 2 columns remove ~99% of soluble proteins, compared to Legacy’s ~97%, and collection volumes are optimised for a high yield of EVs. This means that EV purity, in terms of number of EVs isolated per µg of contaminating protein, is on average 2.6-fold higher on 70 nm Gen 2 columns and 4.6-fold higher on 35 nm Gen 2 columns than their Legacy counterparts (Figure 2). Yield is also increased in Gen 2 columns, at an average of 2.3-fold and 2.5-fold for 35 nm and 70 nm respectively (Figure 2). It’s a win-win!
Smaller pores for smaller particles
New areas of research are constantly emerging. In the last few years, nanoparticles such as supermeres, which are 22-32 nm in size9, have gained attention as a potential source of biomarkers10. In response to the need for isolating these truly tiny particles, we developed a qEV Gen 2 / 20 nm resin to add to our SEC+ product range. This resin recovers over 75% of particles with a 24 nm mean diameter and a higher percentage of particles with a mean size of 50 nm and 70 nm than the Gen 2 / 35 nm Series (Figure 3). Overall, the EV yield is higher for the 20 nm series than either the 35 nm or the 70 nm series resins (Figure 4).
This increased yield from the 20 nm series columns results in lower isolate purity in terms of protein contamination as compared to larger 35 nm and 70 nm pore sizes (Figure 4). However, it is important to note that at least part of this apparent reduction in purity could be due to the co-isolation of supermeres and exomeres, as they are protein-based particles.
With the isolation of these smaller nanoparticles, the problem of lipoprotein contamination may increase, as smaller lipoproteins (such as IDLs and LDLs) will also co-isolate. Thanks to the Gen 2 resin, lipoprotein contamination using our 20 nm series is just as low as using our 35 nm Series (Figure 4).
Reliably high performing
Our production team has optimised a proprietary packing method that produces consistently high-performing columns for every batch. With rigorous quality checks at every stage of the process, you can be confident of a reliable product every time you order. Plus, if your application needs regulatory compliance, we offer made to order GMP-ready columns.
Interested? Find the right column for you: qEV Series Comparison
