Beyond Ultracentrifugation: Embracing the qEV100 for Enhanced Large-Scale EV Isolation Purity and Efficiency

Extracellular Vesicles
About /

The qEV100 is used to isolate extracellular vesicles (EVs) from large sample volumes. How does this size exclusion chromatography column stack up against ultracentrifugation?

Embarking on the daunting task of processing large volumes of conditioned cell culture media or other samples for EV isolation? Avoid the arduousness of ultracentrifugation – a time-consuming process that often yields subpar EV isolates – and forge a better path. Our suggested mode of metaphorical transport? Large-scale size exclusion chromatography (SEC) with our large qEV columns. This article highlights our largest ‘off-the-shelf’ SEC column, the qEV100. Below we will guide you through the critical considerations for large-scale EV isolation, demonstrating why the qEV100 (or possibly one of our even larger custom columns) is the clear winner over ultracentrifugation. Additionally, we'll show you the data which demonstrates how optimising the qEV100 can get the best out of your starting sample.

How does the qEV100 compare to ultracentrifugation for practicality?


Time is valuable, so it is unsurprising that protocol length is an important factor for large-scale EV isolation. Whilst ultracentrifugation protocols often take a full day to execute, the qEV100 collection procedure takes 15 minutes. All in all, with column priming and cleaning, the whole qEV100 protocol takes less than hour to complete. When it comes to time then, there is no contest.

Verdict: Clear qEV100 win for speed of protocol


Here the two methods are pretty evenly tied. qEV100 columns are clearly far cheaper to start using, but have the ongoing cost of buying new columns. Conversely, an ultracentrifuge and everything you need to run it come at a huge initial financial outlay. If this was all there was to it, eventually ultracentrifugation would be cheaper, but the staff time taken to run these day-long protocols and the expensive regular services that the ultracentrifuges require make it dead even.

Verdict: Draw


Aside from maintaining accuracy, the regular servicing of ultracentrifuges is imperative for safety reasons. If an ultracentrifuge is unbalanced or a component fails, ultracentrifuges can be extremely dangerous due to the high g force required for EV isolation. Whole laboratories have been destroyed by ultracentrifuge accidents and in 1992 a man was killed when an ultracentrifuge exploded.

Using a qEV100 column is immeasurably safer.

Verdict: Clear win for the qEV100


Considering the ''Time'' and ''Cost'' sections above...

Verdict: Clear win for the qEV100

How does the qEV100 compare to ultracentrifugation for isolate quality?

EV Yield and Purity

The two big metrics used to assess EV isolation success are yield and purity. Data from the literature suggests that when isolating from larger volumes (e.g., when using cell culture media or biofluids like milk or urine), the yield of EVs is no different when comparing qEV to ultracentrifugation. Purity, however, is different between the two. qEV column isolation results in purer EV isolates (p=0.036), meaning that using qEV100 columns maximise purity without compromising yield. This data was gathered on Legacy qEV column data. With the new Gen 2 qEV columns showing improved purity and the new 20 nm series showing improved yield, you can pick the qEV column that suits your purity and yield requirements.

Verdict: Clear qEV100 win

Figure 1. Data from the literature showing how qEV columns perform in the field for isolation of EVs from a wide range of culture media and large volume biofluid applications.1-6 Statistical analysis is by Mann-Whitney test. n = 6, mean ± standard error.

EV Functionality

One of the biggest reasons to choose qEV columns over ultracentrifugation is the gentle nature of SEC, which results in greater maintenance of EV functionality as compared with UC-isolated EVs.7,8 This is likely in part due to the lack of extreme centrifugal forces such as those used in ultracentrifugation.

Verdict: Clear qEV100 win

Which factors impact upon qEV100 performance?

Whilst the qEV100 was nominally created for a 100 mL loading volume, this isn’t a ‘one-size-fits-all’ rule. For highly concentrated cell culture media, the point of maximum loading volume may be lower. This is why we recommend that you determine the maximum sample loading volume for your sample type. Theoretically, the yield in EVs should double when the sample volume added to the column is doubled. As you can see in Figure 2A, this occurs when doubling the loading of plasma from 25 mL to 50 mL, and between 50 mL and 100 mL (p<0.01). A similar picture is seen with purity (Figure 2B) where increasing the loading volume results in greater purity (p<0.05).

Figure 2. Optimisation of sample loading volume for the qEV100 / 20 nm column. Either 25, 50 or 100 mL of human plasma was run through the qEV100 20 nm series column. EVs and other similarly sized particles were quantified using the Exoid. Protein concentration was analysed using a bicinchoninic acid (BCA) assay. Statistical analysis is by Kruskal-Wallis test with Dunn’s post-hoc test. n = 3-4. Graphs show mean ± standard error. 

Next we sought to show that sample characteristics matter when it comes to choosing the optimal sample loading volume. Here we took 50 mL of plasma and diluted it in another 50 mL of PBS to get a final volume of 100 mL before loading it onto the qEV100. As you can see in Figure 3, there is no difference in the EV yield between the neat or diluted sample. However, there is increased purity for the diluted samples (Figure 3b; p<0.05), demonstrating that 100 mL of a more dilute sample is optimal for the qEV100 than 50 mL of a more concentrated sample. As such, if you have a highly concentrated sample, or have a sample that is less than 100 mL, you may wish to trial dilutions to determine whether you can improve yield and/or purity.

Figure 3. 50 mL of human plasma or 50 mL of human plasma diluted with PBS to 100 mL was run through the qEV100 20 nm series column. EVs and other similarly sized particles were quantified using the Exoid. Protein concentration was analysed using a bicinchoninic acid (BCA) assay. Statistical analysis is by Kruskal-Wallis test with Dunn’s post-hoc test. n = 3-4, mean ± standard error.

The results

As we wrap up this exploration of large-scale EV isolation, it is evident that the qEV100 column sets a new standard in the field. Its swift processing time, combined with high-quality EV isolation and enhanced safety, positions the qEV100 as the optimal choice over the traditional ultracentrifugation. The qEV100 column is designed for a nominal loading volume of 100 mL, but this is not a fixed limit as the optimal volume can be lower for highly concentrated samples. It's recommended to determine the best sample volume for your specific sample type. The qEV100 gives you the flexibility and the opportunity to optimise EV isolation to suit your sample, meaning that you will get the best EV isolate every time.

Whether you want more information on our larger qEV columns or you are interested in learning about our customised large-scale EV isolation columns, our team is here to help.


1. Morozumi M, Izumi H, Shimizu T, et al. Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk. Journal of Dairy Science 2021;104(6):6463-6471, doi:10.3168/jds.2020-19849

2. Veerman RE, Teeuwen L, Czarnewski P, et al. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. Journal of Extracellular Vesicles 2021;10(9), doi:10.1002/jev2.12128

3. Saludas L, Garbayo E, Ruiz-Villalba A, et al. Isolation methods of large and small extracellular vesicles derived from cardiovascular progenitors: A comparative study. European Journal of Pharmaceutics and Biopharmaceutics 2022;170(187-196, doi:10.1016/j.ejpb.2021.12.012

4. Huang LH, Rau CS, Wu SC, et al. Identification and characterization of hADSC-derived exosome proteins from different isolation methods. Journal of Cellular and Molecular Medicine 2021;25(15):7436-7450, doi:10.1111/jcmm.16775

5. Fang X, Chen C, Liu B, et al. A magnetic bead-mediated selective adsorption strategy for extracellular vesicle separation and purification. Acta Biomater 2021;124(336-347,doi:10.1016/j.actbio.2021.02.004

6. Dong L, Zieren RC, Horie K, et al.Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium. Journal of Extracellular Vesicles 2020;10(2), doi:10.1002/jev2.12044

7. Nordin JZ, Lee Y, Vader P, et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine-Nanotechnology Biology and Medicine 2015;11(4):879-883,doi:10.1016/j.nano.2015.01.003

8. Mol EA, Goumans MJ, Doevendans PA, et al. Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation. Nanomedicine-Nanotechnology Biology and Medicine 2017;13(6):2061-2065, doi:10.1016/j.nano.2017.03.011

Related Articles

View All

Subscribe to our newsletter!

Get the latest qEV and TRPS news straight to your inbox.

Check out "Beyond Ultracentrifugation: Embracing the qEV100 for Enhanced Large-Scale EV Isolation Purity and Efficiency " on Izon

No items found.
No items found.
No items found.
No items found.
URL Copied!