Page 78 - Read Online

Gupta et al. Extracell Vesicles Circ Nucleic Acids 2023;4:170-90 https://dx.doi.org/10.20517/evcna.2023.12 Page 172

Size exclusion chromatography
Size exclusion chromatography (SEC) is a widely used chromatography method that is used across various
research fields. SEC serves as a means to separate molecules based on molecular size. Briefly, the sample
which acts as a mobile phase is passed through a porous stationary phase . Smaller particles will be able to
[27]
traverse through more pores as compared to bigger particles, hence resulting in differential elution profiles
where bigger particles will take a shorter path and elute first, followed by smaller vesicles and then non-
exosomal proteins . Pore size and density of the stationary phase are based on the polymer used and can
[26]
be modulated by selecting from the number of gel polymers available such as crosslinked dextran, agarose,
or allyldextran . Due to the limitation of mobile phase volume, which can be subjected to SEC, a pre-
[28]
processing step such as ultrafiltration to concentrate the sample is often performed [26,29,30] . Usually,
precleared cell culture supernatants are subjected to ultrafiltration devices either with dead-end or
tangential flow filtration systems with a molecular weight cut-off ranging from 10 to 1,000 kDa. Dead-end
filtration is more suitable for small-scale applications, while tangential flow filtration is more appropriate for
large-scale production [31,32] . Furthermore, using ultrafiltration with higher molecular weight cut-off values,
relatively pure and intact vesicle preparations can be obtained, hence further improving the efficiency of the
downstream purification system . EVs purified by SEC have better integrity and purity as compared to
[33]
UC-based approaches, as demonstrated by several studies [25,26] . In addition, the scalability of SEC makes it a
promising candidate for EVs purification for GMP production [34-36] . However, since the separation of EVs in
SEC is based on size, the risk of co-elution of VLDL, chylomicrons, and LDL is relatively high in the case of
EV purification from plasma . Furthermore, due to the long processing time and lack of throughput, the
[17]
applicability of SEC in EV-based diagnostics is complicated.

Alternative methods for EV purification
Apart from using density and size, alternative methods have emerged which utilize molecular, biophysical
or biochemical attributes of EVs to segregate EVs from other non-vesicular contaminants and aim to
delineate the heterogeneity of EVs. One way of efficiently capturing EVs in biological samples is to target
specific surface markers known for a particular EV population . Up until now, a wide range of EV markers
[18]
has been identified, which has led to the development of various immune affinity-based approaches for EV
purification. The most commonly used immunoaffinity approaches are directed towards EV-specific
tetraspanins such as CD9, CD81, and CD63 . In addition, some other protein targets have also been used
[37]
[39]
for immunoaffinity purification of EVs, such as MHC antigen and heat shock proteins . Apart from
[38]
protein-based targets, targeting lipids such as phosphatidylserines by Annexin V or targeting
[40]
proteoglycans or other glycocalyx structures by heparin , and lectins [42-44] have been used for affinity-based
[41]
EV purification.
An alternative method employing low-speed centrifugation for EV isolation is precipitation, either by
adding polyethylene glycol or organic solvent (protein organic solvent precipitation technique) [45,46] .
However, these approaches precipitate both vesicular and non-vesicular proteins in the sample. Therefore,
they are not considered EV purification methods but rather methods used to enrich EVs.

Immunoaffinity-based EV purification allows for efficient isolation of pure EVs from complex biological
fluids in a high throughput manner, with minimum user dependency on purity and yield, and has therefore
been deemed ideal for EV-based diagnostic applications [47,48] . For therapeutic purposes, efforts are being
made to increase the scalability and nondestructive retrieval of EVs from the affinity columns [49,50] .

In addition to immunoaffinity, several other technologies have been developed or repurposed for EV
isolation. Amongst others, these include ion-exchange chromatography [51,52] asymmetric flow field
fractionation [53,54] , and microfluidic-based systems [55,56] .