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ABSTRACT Exosomes, a subset of extracellular vesicles (EVs) ranging in size from 30 to 150 nm, are of significant interest for biomedical applications such as diagnostic testing and therapeutics delivery. Biofluids, including urine, blood, and saliva, contain exosomes that carry biomarkers reflective of their host cells. However, isolation of EVs is often a challenge due to their size range, low density, and high hydrophobicity. Isolations can involve long separation times (ultracentrifugation) or result in impure eluates (size exclusion chromatography, polymer‐based precipitation). As an alternative to these methods, this study evaluates the first use of nylon‐6 capillary‐channeled polymer (C‐CP) fiber columns to separate EVs from human urine via a step‐gradient hydrophobic interaction chromatography method. Different from previous efforts using polyester fiber columns for EV separations, nylon‐6 shows potential for increased isolation efficiency, including somewhat higher column loading capacity and more gentle EV elution solvent strength. The efficacy of this approach to EV separation has been determined by scanning electron and transmission microscopy, nanoparticle flow cytometry (NanoFCM), and Bradford protein assays. Electron microscopy showed isolated vesicles of the expected morphology. Nanoparticle flow cytometry determined particle densities of eluates yielding up to 5 × 108particles mL−1, a typical distribution of vesicle sizes in the eluate (60–100 nm), and immunoconfirmation using fluorescent anti‐CD81 antibodies. Bradford assays confirmed that protein concentrations in the EV eluate were significantly reduced (approx. sevenfold) from raw urine. Overall, this approach provides a low‐cost and time‐efficient (< 20 min) column separation to yield urinary EVs of the high purities required for downstream applications, including diagnostic testing and therapeutics.
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Comparison of the separation of proteins of wide‐ranging molecular weight via trilobal polypropylene capillary‐channeled polymer fiber, commercial superficiously porous, and commercial size exclusion columns
Abstract Reversed phase and size‐exclusion chromatography methods are commonly used for protein separations, although they are based on distinctly different principles. Reversed phase methods yield hydrophobicity‐based (loosely‐termed) separation of proteins on porous supports, but tend to be limited to proteins with modest molecular weights based on mass transfer limitations. Alternatively, size‐exclusion provides complementary benefits in the separation of higher mass proteins based on entropic, not enthalpic, processes, but tend to yield limited peak capacities. In this study, microbore columns packed with a novel trilobal polypropylene capillary‐channeled polymer fiber were used in a reversed phase modality for the separation of polypeptides and proteins of molecular weights ranging from 1.4 to 660 kDa. Chromatographic parameters including gradient times, flow rates, and trifluoroacetic acid concentrations in the mobile phase were optimized to maximize resolution and throughput. Following optimization, the performance of the trilobal fiber column was compared to two commercial‐sourced columns, a superficially porous C4‐derivatized silica and size exclusion, both of which are sold specifically for protein separations and operated according to the manufacturer‐specified conditions. In comparison to the commercial columns, the fiber‐based column yielded better separation performance across the entirety of the suite, at much lower cost and shorter separation times.
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- Award ID(s):
- 2107882
- PAR ID:
- 10446184
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Journal of Separation Science
- Volume:
- 45
- Issue:
- 9
- ISSN:
- 1615-9306
- Format(s):
- Medium: X Size: p. 1502-1513
- Size(s):
- p. 1502-1513
- Sponsoring Org:
- National Science Foundation
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