Extracellular Vesicle Purity Enhanced by Gen 2 qEV Columns With Customised Resin

Izon Science has announced the launch of Gen 2, the enhanced range of qEV columns for size-exclusion chromatography-based separation. The new range of qEV columns are made with a high-performance agarose resin, exclusively designed for the isolation of extracellular vesicles (EVs). The resin delivers a more purified EV-containing eluate, compared to the resin used in the existing Legacy range. The release of Gen 2 qEV columns are in line with the need to support the rapidly growing areas of EV research and applications, where sample purity has a huge impact on results downstream.

Update: Following this initial launch, the Gen 2 range has grown. The Gen 2 range now includes the qEVsingle Gen 2, qEVoriginal Gen 2, qEV1, qEV10 Gen 2, qEV100 Gen 2 and qEV2 Gen 2

Remove more protein than before

The resin used in Gen 2 columns enables a greater proportion of protein to be removed from loaded samples, as shown in Figure 1:

Figure 1. Protein present in pooled volumes (2 mL) of a human plasma sample isolated with Izon’s Automatic Fraction Collector, shown as a percentage of the total loaded protein. Protein was measured by bicinchoninic acid assay. Data shown for the qEVoriginal Legacy column and the Gen 2 qEVoriginal column in the 70 nm Series and 35 nm Series (0.5 mL loading volume).

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Increase the EV-to-protein ratio

The enhanced sample purity of Gen 2 columns can also be demonstrated by comparing the EV-to-protein ratio. The number of EVs was obtained via the Exoid, Izon’s tunable resistive pulse sensing (TRPS) nanoparticle measurement system which enables analysis on a particle-by-particle basis. Figure 2 shows separation capabilities in Legacy versus Gen 2 columns:

Figure 2. Number of extracellular vesicles (EVs) per microgram of protein, as measured by tunable resistive pulse sensing and bicinchoninic acid assay, respectively. Data are shown for a human plasma sample separated using Izon’s Automatic Fraction Collector (AFC) and qEVoriginal columns (0.5 mL loading volume) in the 70 nm and 35 nm Series.

The enhanced separation of Gen 2 columns provides more purified EV-containing samples, which is highly desirable for downstream analysis and functional studies.

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Adjust sample composition to suit your downstream analysis

There is also another major benefit to the increased purity seen across purified collection volumes (PCVs). The improved separation provides a new level of control when deciding which PCVs to pool. As shown below, the composition of pooled volumes can be influenced by selecting different PCVs:

Figure 3. A) Elution profile of a human plasma sample separated using a qEVoriginal/35 nm column Gen 2 and Automatic Fraction Collector (AFC). Particle number was measured on the Exoid, protein was measured via bicinchoninic acid assay. Faded bars represent estimated extracellular vesicle (EV) concentration based on calculations and comparisons from various pooled volumes. Different purified collection volumes (PCVs) can be pooled to optimise the sample, depending on the application or analytical method used downstream. Different PCVs can be pooled to prioritise B) EV concentration, C) EV recovery, or D) EV purity. E) The recommended default setting on the AFC prioritises a balance of EV recovery and purity. The user manual provides guidance on when and how the AFC settings need to be adjusted accordingly.

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Further benefits of the Gen 2 qEVoriginal/70 nm column

Following column optimisation, an additional benefit became apparent: the Gen 2 qEVoriginal/70 nm column delivers a higher concentration of EVs, when compared to its Legacy counterpart (the Legacy qEVoriginal/70 nm).

Figure 4. Number of extracellular vesicles (EVs) per mL in a human plasma sample (0.5 mL loading volume) separated on a Legacy qEVoriginal/70 nm column compared to a Gen 2 qEVoriginal/70 nm column collected using the Automatic Fraction Collector (2 mL pooled volume). EV concentration was obtained via the Exoid, Izon’s tunable resistive pulse sensing (TRPS) nanoparticle measurement system which enables analysis on a particle-by-particle basis.

A new column optimised for 1 mL sample loading volumes

The first Gen 2 columns to be released are the qEVoriginal Gen 2, and the qEV1 – a column optimised for a 1 mL loading volume. Both are available now in the existing 35 nm and 70 nm series, which have optimum recovery ranges of 35-350 nm and 70-1000 nm, respectively. The qEV1 is a valuable addition for the many researchers working with lower sample volumes, particularly those with interests in EV-RNA and biomarker discovery and development.

Evolution of the qEV isolation platform

Since the first launch of the qEVoriginal in 2014, the existing range of Legacy columns has expanded to suit different sample loading volumes, including the qEVsingle (2016), qEV2 and qEV10 (2018) and the qEV100 (2019). qEV columns have since been used in studies of diverse sample types including plasma, cell culture supernatant, cerebrospinal fluid, and bronchoalveolar lavage fluid. The introduction of the Automatic Fraction Collector (AFC) in 2019 marked a major shift from labour-intensive manual collection to a rapid and reproducible method that can be scaled up for larger-scale biomarker studies. The release of the Gen 2 columns reflects Izon’s commitment to providing a scalable way forward for EV applications, including EV-diagnostics.

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