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Chromatographia

, Volume 82, Issue 1, pp 415–424 | Cite as

Chromatographic Approaches for Purification and Analytical Characterization of Extracellular Vesicles: Recent Advancements

  • Sara TengattiniEmail author
Review
  • 165 Downloads
Part of the following topical collections:
  1. Rising Stars in Separation Science

Abstract

Extracellular vesicles (EVs) are nanoscale particles released by cells under both physiological and pathological conditions, that mediate intercellular communication by delivering biomolecules (nucleic acids, proteins, and lipids). In the last decade EVs have attracted the interest of scientific community for their potential as diagnostic and therapeutic agents. Despite this interest, analytical issues posed by clinical use of EVs are still relevant and a real standardization of isolation and quality control procedures is far to be defined. A plethora of analytical techniques have been employed for EV isolation and characterization. The aim of this mini review is to provide an overview of the chromatographic approaches that can be applied to intact EVs. Size-exclusion chromatography, affinity chromatography and ion exchange chromatography are the chromatographic modes that have been successfully employed for EV separation. These chromatographic techniques show strengths and weaknesses that are herein discussed, to build a critical picture of potential future role of chromatographic approaches in EV purification and quality control.

Graphical Abstract

Keywords

Extracellular vesicles Extracellular vesicle characterization Extracellular vesicle purification Size-exclusion chromatography Affinity chromatography Ion exchange chromatography 

Introduction

Extracellular vesicles (EVs) are nano-sized membrane-enclosed structures described for the first time by Wolf [1]. EVs contain a variety of biological active molecules, including lipids, proteins, mRNAs and miRNAs and are shed by almost all cell types under physiological and pathological conditions [2, 3]. EVs have different origins and play a fundamental role in many physiological processes, including immune response, cell-to-cell communication, stem cell maintenance and tissue repair after injury, as well as pathological processes including inflammation, viral transfer, tumour invasion, angiogenesis and neurodegenerative disorders [4, 5, 6].

Three major types of EVs, differentiated based on their biogenesis, have been described: apoptotic bodies (> 1000 nm), microvesicles (100–1000 nm) and exosomes (30–100 nm). Apoptotic bodies are released by apoptotic cells, microvesicles are directly formed from the plasma membrane, while exosomes are originated from the intraluminal budding of multivesicular bodies (MVBs) of most cell types (including dendritic cells, endothelial cells and tumour cells) [7, 8].

Since EVs are endogenous messengers and their composition (lipids, proteins and nucleic acids) reflects the content of secreting cells, they can influence physiological and pathological processes and be considered as representative of the state of cellular health. For this reason, EVs have attracted consideration as potential diagnostic tools [3, 9, 10]. EV diagnostic potential is extremely attractive since they can be detected in biological fluids (including blood, saliva, urine and breast milk) without invasive surgery [11].

A very promising and widely investigated potential of EVs concerns the use of tumour-associated vesicles as cancer diagnostics. Scientific evidence suggests that EVs could represent an extremely useful and accessible source of cancer biomarkers with prognostic and predictive value. Hence, EVs might find application in early stage diagnosis and disease progression monitoring [12, 13, 14, 15].

EVs have also a considerable potential as therapeutic agents. Taking advantage of their intrinsic targeting capacity, EVs can be used as tissue- and organ-specific drug delivery systems (DDSs) to increase the delivery efficiency of drugs (such as anticancer drugs, therapeutic microRNAs, small interfering RNAs (siRNAs) and proteins) and reduce the side effects [11, 16, 17]. In addition to their high biocompatibility and safety, low immunogenicity and ability to easily traverse biological barriers, EVs can also be modified and engineered to enhance their delivery and pharmacokinetic properties [2].

Considering their potential as diagnostics and therapeutics, scientific interest in EVs has markedly raised in the last 10 years as demonstrated by the exponential increase in the number of scientific publications [13].

Clinical application of EVs requires a dual support from analytical techniques. On one hand a major challenge in this field is finding scalable isolation and purification methods that yield intact functional EVs [18, 19, 20]. On the other hand, since methods able to isolate EVs from biological fluids quantitatively and without impurities have not yet been described, suitable analytical tools for the qualitative and quantitative characterization of EV samples are needed [21, 22, 23]. Despite the high interest in the clinical exploitation of EVs, standardization of analytical techniques used for EV characterization are yet to be performed and quality control procedures are far from being clearly defined [24].

A plethora of analytical methods, including ultracentrifugation (UC), ultrafiltration (UF) and immunoprecipitation, have been employed to isolate EVs [25, 26, 27, 28]. However, there is no ideal isolation technique even if UC is considered the gold standard technique and a combination of methods are used to obtain EVs with good purity and integrity for clinical applications. Size-exclusion chromatography (SEC) can be considered a promising alternative, together with affinity chromatography (AC) and ion exchange chromatography (IEX), as simple and efficient method for the purification of EVs in standardized conditions [22].

Optical and non-optical methods, such as transmission electron microscopy (TEM), atomic force microscopy (AFM), nanoparticle tracking analysis (NTA) and flow cytometry, have been used for the analysis of the composition and morphology of EV populations [21, 25, 27, 29, 30]. In the last years, cryo-electron microscopy (Cryo-EM) has emerged as ideal technique to reveal detailed structural features in EVs, allowing to determine morphological heterogeneity [25].

Western blot analysis, proteomics, lipidomics, glycomics and RNA and/or DNA sequencing are also needed to profile EV constituents. The structural characterization can improve the level of molecular details on EVs isolated from different biological sources and isolation protocols [31]. Chromatographic approaches, often hyphenated to mass spectrometry, suitable for the characterization of EV cargo composition have been recently reviewed by other authors [32, 33]. Instead, this mini review wants to be an overview of the more recent chromatographic methods applied to the purification, characterization, and quality control of intact EVs. As reported in Fig. 1, the number of publications that deal with “extracellular vesicles” and “chromatography” is exponentially growing in the last years (from 1990 to 2017). Most significant recent publications are herein discussed to show potentialities and limitations of the different chromatographic modes.

Fig. 1

Histogram of the number of publications regarding chromatographic analysis of EVs per year over the past 28 years. The histogram was generated in PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) using the keywords “Extracellular vesicles” and “Chromatography”

Chromatographic Approaches for Intact EV Isolation and Characterization

Chromatographic approaches based on SEC, AC and IEX have been applied to the isolation and characterization of EVs to be used as disease markers or as carriers for drug delivery. These three techniques take advantage of different EV properties, that are, respectively, size, surface marker presence and surface charge (Fig. 2). The advantages and disadvantages of each mode are summarized in Table 1, while selected examples are discussed below.

Fig. 2

Illustration of the separation mechanism of EVs in a size-exclusion chromatography, b affinity chromatography and c anion exchange chromatography

Table 1

Comparison of the three different chromatographic modes used for EV isolation and characterization

Chromatographic mode

Application

Advantages

Disadvantages

References

SEC

Isolation/purification

Reduced contamination by low MW impurities

Rapid (min)

No specialized equipment required

Intact and functional vesicles

May require sample pre-concentration

May require concentration of eluted sample

Contamination by other particles of similar size

Low quantity recovered

[18, 34, 35, 37, 38, 39, 40, 41, 42]

Analytical characterization/quantification

Rapid (min)

No specialized equipment required

Selectivity can be modulated modifying pore dimension

Low selectivity (large size differences are required)

Low ability to discriminate among EV populations

[44, 45]

AC

Isolation/purification

High specificity of interaction

Selective enrichment of EV populations

Easily scalable

Low Abs purity and stability

Low availability of specific Abs

Non-specific binding

Binding not easily reversed

EV functionality can be compromised

[32, 56, 57]

Analytical characterization/quantification

High specificity of interaction

Easy detection of specific markers

Characterization of EV composition

Low Abs purity and availability

Non-specific binding

[32, 56]

AIEX

Isolation/purification

High purity

Non-specific binding

Low ability to discriminate among EV populations

[58, 59, 60]

Size-Exclusion Chromatography

In SEC the separation is based on molecule’s physical size in solution. The elution order is based on the hydrodynamic volume of the analytes and generally reflects their molecular weight. High molecular size analytes elute first because they are excluded from the pores of stationary phase matrix. On the contrary, smaller molecules can access pore within the matrix particles and thus permeate a larger accessible volume within the column, eluting later (Fig. 2a).

SEC has become one of the methods of choice for EV isolation, as it allows to achieve a high-quality purification in short time, without specialized equipment and starting from highly complex physiological fluids, such as serum or plasma [34, 35, 36, 37, 38]. When EVs are produced for therapeutic application, a suitable purification method must be able to guarantee good purity, but preserving vesicle structure and functionality. The reduction of isolated EV functionality and therefore of their effectiveness as therapeutic agents is one of the main aspect limiting the success of most used purification protocols, such as UC [24]. Recently, Mol et al. [18] demonstrated that purification by SEC led to the isolation of samples with higher degree of intact and functional EVs in comparison with the traditional isolation by differential UC, avoiding aggregation and fusion phenomena. A comparison between the same two techniques was made by Baranyai et al. [39] in the purification of exosomes from blood plasma, with the finding that SEC resulted in reduced albumin contamination and in a final product of higher quality. Likewise, Gámez-Valero et al. [40] compared SEC-isolated EVs with vesicles obtained by precipitation with polyethylene glycol (PEG) or by PRotein Organic Solvent Precipitation (PROSPR). Morphological and functional analysis showed that SEC purification resulted in a lower amount of contaminant plasma proteins and in the preservation of vesicular structure and conformation.

An important aspect that has to be considered in the development of EV purification methods is the ability to support the large-scale production required for therapeutic applications [24]. If the advantages of SEC in EV purification are several and undeniable, the main disadvantage of this technique is the low yield of recovery, that makes the scale-up challenging [40]. However, successful scale-up attempts start to appear in the literature [41, 42]. In 2017, Corso et al. [42] developed an easily scalable method based on the use of commercially available bind-elute size-exclusion chromatography (BE-SEC) columns. These columns are made of beads able to trap molecules smaller than 700 kDa via hydrophobic and ionic interactions with the positively charged octylamine ligands present inside the bead core. Larger particles, unable to permeate inside the beads, pass through. EVs, that are larger than 700 kDa, are thus directly eluted in the solvent front, while soluble proteins, RNAs and small sized impurities remain trapped. The investigators demonstrated that the developed BE-SEC method allowed to purify functional EVs in a time-efficient manner, with yields ranging from 70 to 80% and purity comparable to the gold standard methods in the field. Moreover, they proved the ability of their system to purify EVs from volumes of cell culture medium larger than laboratory scale (from 50 up to 200 mL). The positive results demonstrated that BE-SEC could be considered a promising approach for large-scale production of EVs for therapeutic purposes.

Parallel to the consolidated application as preparative tool, SEC has a well-established potential as qualitative and quantitative analytical technique. In biopharmaceutical analysis, it is considered a standard method for the quality control of therapeutic proteins and macromolecules [43]. However, analytical application of SEC to EV characterization is just in its infancy. In 2017, Huang and He [44] took advantage from the qualitative potential of SEC proposing a simple SEC method to investigate the purity of EVs isolated from cancer and endothelial cells. The SEC-UV method demonstrated to be able to properly resolve EV particles from water-soluble impurities of small size, mainly proteins, present in the samples. For this application, the authors selected a Superose 12 10/300 GL column with an exclusion limit of 2 × 106 Da, that allowed to achieve a baseline resolution of small-size impurities from small EVs (sEVs) with size less than 200 nm, and hypothesized that the use of columns with higher exclusion limit might permit to resolve small EVs from larger particles, but losing resolution from small-size molecules.

SEC can be successfully employed also for quantitative characterization of EV particles. Xu et al. [45] in 2016 developed a rapid and inexpensive method based on SEC coupled with fluorescence detection (SEC-FD) for sEV quantification. The authors tested several commercially available SEC matrices to prepare an in-house SEC column, and finally selected Sepharose CL-4B. The SEC-FD protocol was first tested and validated in the analysis of commercially available 100 nm diameter liposomes, showing good selectivity (in the separation from albumin, assumed as model of contaminant macromolecules), linearity, sensibility and repeatability. The method was then applied to the quantification of sEVs in a real sample, allowing the sensitive and easy monitoring of the number of sEVs secreted from the human B-lymphoblastoid cell line TK6, cultured in serum free medium for a period from 1 to 48 h.

However, apart from a few number of examples as those here reported, the main application of SEC in EV field remains their isolation and purification during the production process, while the use of SEC for the analytical characterization is still scarcely explored. A class of analytes that has a high structural similarity with EVs in terms of dimensions and physical–chemical properties is represented by virus and virus-like particles (VLPs). In the characterization of these complex and high-molecular weight entities we can find further evidence of the analytical potential of SEC [46, 47, 48, 49].

In 2016 Vajda et al. [48] developed a SEC method for size distribution analysis of influenza virus particles, demonstrating the ability of SEC to discriminate virus particle monomers from aggregates and fragments and to offer a detailed picture of the virus size distribution, which is an important quality parameter. Moreover, Steppert et al. [49] developed a SEC method for the quantification of enveloped VLPs in various sample matrixes and at different purity stages, for a rapid and user friendly in-process control. In this work SEC separation was coupled to UV detention and, alternatively, to multi-angle light scattering (MALS) detention. The use of MALS allowed direct particle quantification with no need of a calibration curve, which is mandatory when UV detention is employed.

The selected examples on EVs, viruses and VLPs clearly demonstrate that it is possible to achieve satisfactory chromatographic performances in membrane-enclosed particle analysis.

A key step to developing a robust SEC analytical method is the selection of proper stationary and mobile phases. Mobile phase “needs” to have a sufficient ionic strength to prevent particles from secondary interactions with the stationary phase. Obviously, the column selection has the greater influence on method performance, since pore dimension distribution determines chromatographic selectivity and resolution. Table 2 summarizes SEC stationary phases that have been applied to the isolation and/or characterization of EVs, considering the scientific papers selected for this mini review. Because of the structural similarity, also the examples concerning the chromatographic analysis of viruses and/or VLPs are reported. All the discussed aspects indicate that SEC can be considered a suitable technique that could fulfil the urgent demand of tools for EV characterization and quality control prior to their clinical application.

Table 2

SEC columns used for EVs, viruses and/or VLPs purification and analysis in the considered papers, their properties and applications

Manufacturer

Column

Stationary phase material

Pore size (nm)

Analyte

Application

References

GE Healthcare

Column packed in-house with Sepharose 2B particles

Agarose

 

Exosomes

Isolation from plasma

[39]

Column packed in-house with Sepharose CL-2B and 4B particles

Cross-linked agarose

 

Exosomes, liposomes, EVs

Isolation from plasma and cell culture

Quantification of EVs secreted from cells

[36, 37, 39, 40, 45]

HiPrep Sephacryl S-400

Dextran/bisacrylamide

 

EVs

Isolation from cell culture

[18, 39]

Superdex 200 column

Agarose and dextran

 

EVs

Large-scale purification from cell culture

[41]

Superose 12 10/300 GL

Agarose

 

EVs

Impurity and contaminant detection

[44]

Izon Science

qEV

 

~ 75

Exosomes, exosome-like vesicles

Isolation from various starting materials (plasma, biological fluids, etc.)

[35, 38]

Tosoh Bioscience

TSKgel G5000 PWxl

TSKgel G6000PW

TSKgel G6000 PWxl TSKgel G-DNA PW

Cross-linked hydroxylated polymethacrylate

100

> 100

> 100

> 100

Viruses

VLPs

Characterization

Quantification in various sample matrixes

Aggregate and fragment detection and quantification

Size distribution analysis

Stability studies

[46, 47, 48, 49]

Waters Corporation

ACQUITY UPLC Protein BEH450

Hybrid material (silica and polymeric) with diol coating

45

VLPs

Characterization

Aggregate detection and quantification

Size distribution analysis

Stability studies

[47]

Sepax Technologies

SRT SEC-1000

Hydrophilic neutral films chemically bonded on silica

100

VLPs

Characterization

Aggregate detection and quantification

Size distribution analysis

Stability studies

[47]

Affinity Chromatography

AC exploits highly specific interactions for isolating target molecules within complex biological mixtures. EVs expose on their outer surface molecules that can be targeted by their specific interaction with ligands immobilized on the stationary phase (Fig. 2b). Generally, the targeted molecules are protein markers (such as tetraspanins, heat-shock proteins, Tsg101 and annexin V) and the affinity ligands are antibodies (Abs) able to specifically recognize these proteins [32]. Other affinity ligands have been considered such as peptides which bind to heat-shock proteins [50] or heparin, that was proved to be involved in specific interactions with different proteins exposed by EVs [51]. An interesting class of affinity ligands useful in EV characterization are lectins, which are carbohydrate-binding proteins able to specifically recognize glycans present on EV surface [52]. The presence of glycoproteins on EV surface is well established and it is known to be involved in many functions, from their biogenesis to their uptake by recipient cells [53, 54]. Lectin-based affinity methods have thus been developed not only to isolate EVs [52], but also to profile and characterize EV glycosylation [55].

Based on the diffusion of affinity target, the resulting method can be specific for a class of vesicles or for EVs in general [32].

Even if, theoretically, affinity methods should result in highly pure EV preparation due to the extreme specificity of the antigen–Abs interaction, the potential of this chromatographic mode can be limited by the critical quality and stability of Abs and the occurrence of non-specific binding, often resulting in low-purity EV samples. In addition, in general, immuno-affinity methods are based on binding interactions that are not easily reversible. The elution of the retained EVs can thus compromise their integrity and functionality. Finally, only few EV-specific Abs are available. For all these reasons, affinity methods are generally more suitable for analytical applications, in which the elution of intact, sufficiently pure and functional EVs is not required and the main goal is EV (or a particular class of EV) detection and quantification [32].

Nevertheless, in the last years same significant examples of EV isolation by affinity methods appeared in literature. Among these, Yoshida et al. [56] developed an affinity method based on the interaction between phosphatidylserine (PS) residues, present on EV outer lipid bilayer, and TIM4, a protein highly expressed in macrophages that selectively binds PS in a specific and calcium-dependent manner. EV elution was simply achieved by chelating calcium with EDTA and resulted in pure and intact vesicles. The reported method involved the use of magnetic beads, but the considered interaction could be successfully applied in a chromatographic system.

An affinity chromatography-based method was, instead, the one developed in 2018 by Hung et al. [57] for the selective isolation of a specific EV subset starting from a heterogeneous EV population. In this work, the authors fused an affinity tag, the FLAG tag (DYKDDDDK), to the extra-vesicular terminus of the late endosomal transmembrane protein Lamp2b, which is incorporated into exosomes. After the transfection of EV-producing cells with this affinity-tagged version of the protein, tag-protein was display on the surface of exosomes, that were thus specifically isolated using an anti-FLAG affinity column. Selectively enriched and functional tagged EVs were obtained. If anti-FLAG affinity chromatography is applicable only at a research-scale EV isolation, the potential of this approach is in the possibility to successfully readapt the method to other affinity interactions and affinity chromatographic systems, enabling a larger scale EV production for therapeutic and diagnostic applications.

Moreover, it has to be underlined that the ability of AC to discriminate among different EV classes and populations is one of the most interesting aspect of this approach. In fact, while EV heterogeneity is well documented, methods for separating specific EV subclasses are missing due to the overlapped properties of different EV types in terms of size and density [57].

Ion Exchange Chromatography

In ion exchange chromatography (IEX) the analyte retention is based on ionic interactions with the negatively or positively charged stationary phase. Taking advantage of the net negative charge found on EV surface, anion exchange chromatography (AIEX) has been applied to their isolation [58, 59, 60]. EVs strongly interact with the positively charged chromatographic matrices used in AIEX, allowing their isolation from complex mixtures such as cell cultures (Fig. 2c). Elution is driven by the increase in the ionic strength of the mobile phases. Recently, Kosanovic et al. [59] compared AIEX to the sucrose density gradient (SDG) centrifugation method in the purification of EVs isolated from human amniotic fluid (AF) and demonstrated that AIEX enabled efficient separation of negatively charged EVs from other differently charged molecules, resulting in highly pure preparations.

Moreover, Heath et al. [60] employed an AIEX monolithic column for the isolation of EVs from different cell lines and compared them to EVs isolated by UC and tangential flow filtration (TFF) in terms of yield, size, morphology, and contaminant presence. The authors demonstrated that AIEX allowed a similar, or even slightly better in terms of purity, isolation of EVs in comparison to UC, while TFF resulted in significant higher contamination and in the partial loss of vesicle integrity (Fig. 3).

Fig. 3

Comparison of marker content and size distribution profiles of AIEX-isolated EVs with EVs obtained by UC and TFF. a Western blot for EV markers (CD63, CD81, ALIX, TSG101) and potential preparation contaminants (BSA, Calnexin) in cell lysates, EVs isolated from clarified media and a media-only control (10% EV depleted FBS-containing media that had not been exposed to cells) by UC, AIEX and TFF n = 3. bd NTA data showed the size distribution (data shown for 1–499 nM only, with a 10 nm bin width), average concentration and average size of EVs isolated by each technique n = 3. Error bars ± SEM. e Cryo-electron microscopy images of EVs after isolation by AIEX, UC and TFF. White arrows indicate EVs. Black arrows indicate lipid droplets in TFF sample. Asterisks indicate areas showing protein contamination n = 3. Scale bar = 50 nm. From [60]

These works pave the way to the use of AIEX for EV purification.

Conclusions

EVs are novel promising diagnostic and therapeutic agents in many application fields, particularly in cancer treatment. However, parallel to the growing interest in EV functions and the increasing evidence of their great potential, standardized methods for their characterization and quality assurance on analytical scale and for their isolation and purification on preparative scale are still missing. A plethora of different techniques have been employed for EV characterization, targeting different EV properties (such as size, morphology, marker presence, cargo composition), while for their purification, at present, the gold standard is based on UC methods. Despite the analytical efforts, the main goal to obtain functional, highly pure and well-characterized EV preparations has still to be address.

Chromatographic methods might represent promising approaches for simple, high-throughput and cost-effective purification and analysis of EVs.

Among different chromatographic modes, SEC is the most consolidated approach for EV analysis, and it is candidate to have an increasing role both in EV purification and quality control. AC, as for other affinity-based methods, can be successfully employed for EV characterization, while its affirmation as preparative tool is still ongoing. Differently, AIEX is intended to have an increasing importance in production processes, as isolation and purification approach, but shows a low potential as analytical tool.

For therapeutic application of EVs, additional efforts are necessary to scale-up chromatography-based methods from laboratory to productive scale, while for their use as diagnostic markers, chromatographic system requires to be scaled down, for instance to microfluidic systems [19, 20, 61].

Notes

Compliance with Ethical Standards

Conflict of interest

Sara Tengattini declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by the author.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Drug SciencesUniversity of PaviaPaviaItaly

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