Unlike conventional filtration in which the solution is passed through a size-exclusion membrane once and “head-on”, CFF recycles the input solution and passes it parallel to the filtration membrane ( Figure 1(a)). Hence, we sought a large volume method that removed serum contaminants without subjecting the EV to physical or chemical forces.ĬFF, also referred to as tangential-flow filtration, is a technique of nanofiltration that is used for the purification of biomolecules such as therapeutic antibodies, viruses or antibiotics. Serum proteins in particular have been shown to alter the biological activity of EVs. These contaminants can lead to EV circulating DNA and long RNAs also precipitate under these conditions, making it difficult to assign a biological phenotype to any one molecular entity. Finally, use of crowding reagents, particularly on fluids such as serum/plasma, tend to co-precipitate large proteins and ribonucleic acid:protein complexes such as Ago-complexed miRNA. A separate first step isolation method is SEC systems, which allow for little more than 20 mL input. The role of extracellular vesicles (EVs) has received considerable attention in recent years, in particular the class of EVs with a diameter 100,000 g) onto EV, risking lysis and denaturation. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Through this combination, EVs loss is kept to a minimum. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. This information could be used to predict and optimize the purification of large biomolecules and bioparticle in route to the establishment of more effective downstream processes.Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. This study provides qualitative and quantitative information about Capto™ Core 700 resin. The DBC 10% at 2 min residence time are 24 and 2 mg/mL for BSA and Tg, respectively. Effective pore diffusivities values in the core are 1.6 × 10 −7 and 0.16 × 10 −7 cm 2/s for BSA and Tg, respectively. For both proteins, the effective pore diffusivity in the core is smaller than in the shell due to additional hindrance by bound protein in the core area. The addition of 500 mM NaCl reduces the binding capacity by less than 50%, showing the ability of the resin to operate at high salt conditions. Both proteins present highly favorable binding isotherms with maximum binding capacities of 55 and 105 mg/mL of total bead volume for BSA and Tg, respectively. The resin average bead size is 90.7 μm with a range of 50–130 μm, the shell thickness is 4.18 µm with a range of 3–6 µm and a standard deviation of 0.55 µm, and the pore radius, obtained by inverse size exclusion chromatography, is 50.4 ± 1.3 nm. The functionalized adsorbing core and the inert shell have the same fibrous structure typical of agarose-based beads. The present study aims to characterize the structure and functional properties of this resin using bovine serum albumin (BSA, Mr~65 kDa) and thyroglobulin (Tg, Mr~660 kDa) as model impurity proteins. Capto™ Core 700 is a core-shell chromatographic support with an adsorbing core contained within an inert shell layer designed to purify larger biomolecules and bioparticles in a flow-through mode.
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