Rapid method for the high purity isolation of bovine milk-derived extracellular vesicles via polyester (PET) capillary-channeled polymer (C-CP) fiber columns.

Abstract

The use of raw bovine milk as a source of extracellular vesicles (EVs) has gained in interest for therapeutic applications due to its low cost, accessibility, low immunogenicity, and potential for oral delivery. To address the need to achieve higher throughput isolation of high-quality EVs, high performance liquid chromatography (HPLC) using a hydrophobic interaction chromatography (HIC) capture/elution program is performed on microbore, capillary-channeled polymer (C-CP) fiber columns. Methods: Bovine milk is first skimmed to reduce the fat-laden matrix, followed by treatment using acetic acid (Ac) to precipitate casein micelles before isolation of milk-derived EVs (MDEVs) via HPLC using an HIC process modality with PET C-CP fiber columns. The treated milk is introduced to the column under EV binding conditions, where an ionic solvent with a small amount of organic modifier causes salts, small molecules, and proteinaceous species to pass through unretained while retaining the EVs on-column. The target EVs are eluted by decreasing the ionic strength of the solvent and increasing the elution strength. Results: EVs isolated using PET C-CP fiber columns demonstrate the removal of >95% of matrix-related concomitant species and yield particle densities on the order of 10¹¹ particles mL^-1 in 20 min. Validation of the success of the separation is demonstrated through response curves, nanoflow cytometry, transmission electron microscopy, and protein assays in accordance with MISEV guidelines. Conclusions: A rapid approach to the high yield isolation of high-quality MDEVs via microbore-scale PET C-CP fiber columns is presented here. Each separation yields MDEVs on the order of 4 x 10¹¹ particles mL^-1, in 20-min at a cost of <$5 per column. Paths forward to greater EV throughput and yields are currently under development.