Rapid Separation of Blood Plasma Exosomes from Low-Density Lipoproteins via a Hydrophobic Interaction Chromatography Method on a Polyester Capillary-Channeled Polymer Fiber Phase

Abstract Exosomes are membrane-bound, cell-secreted vesicles, with sizes ranging from 30 to 150 nm. Exosomes in blood plasma have become proposed targets as measurable indicators of disease conditions. Current methods for plasma-based exosome isolation are time-consuming, complex, and have high operational costs. One of the most commonly reported shortcomings of current isolation protocols is the co-extraction of lipoproteins (e.g. low-density lipoproteins, LDLs) with the target exosomes. This report describes the use of a rapid, single-operation hydrophobic interaction chromatography (HIC) procedure on a polyester (PET) capillary-channeled polymer (C-CP) fiber column, demonstrating the ability to efficiently purify exosomes. The method has previously been demonstrated for isolation of exosomes from diverse biological matrices, but questions were raised about the potential co-elution of LDLs. In the method described herein, a step-gradient procedure sequentially elutes spiked lipoproteins and blood plasma-originating exosomes in 10 minutes, with the LDLs excluded from the desired exosome fraction. Mass spectrometry (MS) was used to characterize an impurity in the primary LDL material, identifying the presence of exosomal material. Transmission electron microscopy (TEM) and an enzyme-linked immunosorbent assay (ELISA) were used to identify the various elution components. The method serves both as a rapid means of high purity exosome isolation as well as a screening tool for the purity of LDL samples with respect to extracellular vesicles.