Which qEV Column Delivers the Purest EV Isolate?

Extracellular Vesicles
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If you want the purest EV isolate which qEV column should you pick?

Different extracellular vesicle (EV) applications and stages of research require different EV isolate parameters to be prioritised. For instance, you might be after the most concentrated EV sample possible. Or, you might be looking to maximise EV recovery, while accepting a slightly higher degree of protein contamination. For many, though, purity is of most importance. It is impossible to assign a functional impact to EVs if there are contaminants in the EV isolate. Equally, for diagnostics or exploratory ‘omics experiments the presence of contaminants might lower the signal-to-noise ratio and make biomarkers harder to detect. For therapeutics too, purity is essential, as delivering contaminants to patients could result in unforeseen side effects. If you have purity on the brain, then this article is for you.

Here, we take a look at our qEV columns and compare them for their ability to deliver on purity, so that you can make an informed decision on column choice for your research. While we used the qEVoriginal for these comparisons – the most popular column size – the findings are applicable to all column sizes using the respective resins.

Which qEV column has the lowest protein contamination?

In the EV field the degree of protein contamination is colloquially referred to as ‘purity’. Calculated by dividing the EV concentration by the protein concentration, EV purity gives a value of EVs per µg of protein. To determine EV concentration in the purified collection volume (PCV; the EV-rich isolate) from our columns, we used the Exoid to measure EVs and similarly sized particles and set a lower cutoff of 60 nm for analysis. We use this cutoff to ensure that we can compare like for like as much as possible.

For this we compared our Gen 2 columns to their Legacy counterparts in both the 35 nm and 70 nm Series. It is worth noting that the Legacy columns are being phased out across 2023, and so may not be available depending on when you are reading this article. Still though, you or studies that you have read may have used the Legacy columns and you would logically want to know how they measure up to our Gen 2 columns.

In a nutshell, the ability of Gen 2 columns to deliver a purer EV isolate was the main reason for developing the Gen 2 range in the first place. EV isolates from Gen 2 columns for both the 35 nm and the 70 nm Series are significantly purer than what can be obtained with their Legacy counterparts (Figure 1). Compared to the Legacy range, EV isolates from the Gen 2 / 35 nm columns and Gen 2 / 70 nm columns are 2.9-fold and 2.3-fold purer, respectively.

Graphs showing that Gen 2 columns are purer than Legacy columns for both the 35 nm and 70 nm series
Figure 1. A comparison between the purity of EV isolates from qEV Legacy and Gen 2 columns. Purity was calculated by measuring the concentration of particles >60 nm using tunable resistive pulse sensing (TRPS) on an Exoid in the purified collection volume(PCV) and dividing this by the concentration of protein in the PCV as determined by a protein assay. (A) EV purity in PCVs isolated using the 35 nm Series (n = 11 for Legacy, n = 15 for Gen 2). (B) EV purity in PCVs isolated using the 70 nm Series (n = 10 for Legacy, n = 15 for Gen 2). All data were normally distributed. Data are presented as mean ±standard error. Statistical analysis was t test. * p < 0.05, ** p < 0.01

Do Legacy or Gen 2 columns isolate EVs with the lowest lipoprotein contamination?  

To assess the efficiency of lipoprotein removal, we conducted ELISAs for two of the most commonly analysed lipoprotein markers: ApoA1 and ApoB. ApoA1 is a marker of high-density lipoproteins (HDL), the smallest type of lipoprotein. The amount of ApoA1 in EV isolates from our Gen 2 columns   is less than 0.005% of the total ApoA1 from the pre-isolated plasma samples. This is such effective removal of ApoA1 that we expect that the levels of HDL contamination in qEV Gen 2 column isolates to be incredibly low. This is not unexpected as HDL particles are less than 15 nm in diameter, and neither the 35 nm nor 70 nm columns should isolate particles of that size within the PCV.

When it comes to ApoB the story is a little different. This is because ApoB is a marker of intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and very low density lipoproteins (VLDL) which all overlap with the size of EVs. As such, they are likely to be co-isolated with EVs more than HDL. As you can see in Figure 2, the Gen 2 versions of both the 35 nm and 70 nm columns significantly improved the depletion of ApoB – and, thereby, likely the IDL, LDL and VLDL lipoprotein contamination – in EV isolates. The lowest levels of ApoB came with the Gen 2 / 70 nm columns which have significantly lower ApoB levels than the Gen 2 / 35 nm columns (p<0.05).

ApoB level comparisons between Legacy and Gen 2 columns
Figure 2. The proportion of ApoB measured in the EV isolate (% of concentration in loaded sample)  of Legacy and Gen 2 qEV columns. The levels  of total ApoB were measured by ELISA in raw plasma samples or in the EV isolate (PCV) generated by running that sample through a qEV Legacy or Gen 2 / 70 nm column. There was an n = 6 where each of 3 columns were used twice. Data were normally distributed and statistical analysis was by t test. Data are presented as mean ± standard error. * p < 0.05, ** p < 0.01

So, which qEV column isolates the purest EVs?

Reviewing all the evidence shown here, the clear winner is the qEV Gen 2 / 70 nm Series. Whilst its purity in terms of protein contamination is on par with the qEV Gen 2 / 35 nm Series, it really comes into its own in removing lipoprotein contamination from EVs. In these tests, the qEV Gen 2 / 70 nm Series removed 99.4% of protein, 99.9% of ApoA1 (HDL) and 99% of ApoB (IDL, LDL and VLDL). We may be biased, but we think that’s really good. If you want pure EVs, the data presented here shows that the qEV Gen 2 / 70 nm Series is a safe bet.

Sound good? Read more about our columns.


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