Heparin affinity chromatography for extracellular vesicle purification and fractionation

The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. A team led by researchers at University College London evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Their findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. The researchers found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, they detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications.

Experimental overview

This study describes two main sets of experiments. Firstly, they compared heparin affinity chromatography (HAC), using a linear elution gradient, to size-exclusion chromatography to assess the potential of HAC to capture extracellular vesicles (EVs). Secondly, they replaced the linear elution gradient with a step gradient in the HAC protocol and evaluated the purification performance and ability to fractionate EVs. TFF: tangential flow filtration material.