Search for: All records

Abstract Extracellular vesicles (EVs) are released by all cell types into the extracellular environment. A subset of EVs, known as exosomes, range in size from 30 to 200 nm and are of biochemical interest due to their function as vehicles of intercellular communication. Their ability to transport proteinaceous species and genetic material at the cellular level makes them prime candidates as vectors in gene therapies. Focusing on biotherapeutics, bovine milk–derived extracellular vesicles (MDEVs) hold particular promise as an alternative to other exosome sources for therapeutics delivery. Bovine milk poses unique challenges due to the complex colloidal matrix, composed predominantly of fats and proteins like casein, which form micelles that exhibit exosome-like characteristics, specifically size and density. When faced with complex matrices like milk, conventional size/density-based isolation methods including ultracentrifugation and size exclusion chromatography struggle to provide high purity yields on practical time and cost scales. When paired with a stepwise hydrophobic interaction chromatography (HIC) gradient, polyester (PET) capillary-channeled polymer (C-CP) fibers in column and spin-down tips formats have been used effectively to isolate exosomes from highly diverse sources. Here, PET C-CP fiber columns are demonstrated to isolate MDEVs from pre-treated raw milk, yielding concentrations of 1.5 × 10¹⁰particles mL⁻¹with purities of ~2 × 10¹⁰EVs µg⁻¹protein in less than 20 min. The efficacy of the isolation process is verified by a suite of characterization methods. Implementing PET C-CP fiber columns for MDEV isolation addresses the challenges associated with conventional isolation methods, holding promise for scale-up towards therapeutic applications. 

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. 

Abstract Extracellular vesicles (EVs) have garnered much interest due to their fundamental role in intracellular communication and their potential utility in clinical diagnostics and as biotherapeutic vectors. Of particular relevance is the subset of EVs referred to as exosomes, ranging in size from 30 to 150 nm, which contain incredible amounts of information about their cell of origin, which can be used to track the progress of disease. As a complementary action, exosomes can be engineered with therapeutic cargo to selectively target diseases. At present, the lack of highly efficient methods of isolation/purification of exosomes from diverse biofluids, plants, and cell cultures is a major bottleneck in the fundamental biochemistry, clinical analysis, and therapeutic applications. Equally impactful, the lack of effective in-line means of detection/characterization of isolate populations, including concentration and sizing, is limiting in the applications. The method presented here couples hydrophobic interaction chromatography (HIC) performed on polyester capillary-channeled polymer (C-CP) fiber columns followed by in-line optical absorbance and multi-angle light scattering (MALS) detection for the isolation and characterization of EVs, in this case present in the supernatant of Chinese hamster ovary (CHO) cell cultures. Excellent correlation was observed between the determined particle concentrations for the two detection methods. C-CP fiber columns provide a low-cost platform (< $5 per column) for the isolation of exosomes in a 15-min workflow, with complementary absorbance and MALS detection providing very high-quality particle concentration and sizing information. 

Abstract Exosomes, a subset of extracellular vesicles (EVs, 30–200‐nm diameter), serve as biomolecular snapshots of their cell of origin and vehicles for intercellular communication, playing roles in biological processes, including homeostasis maintenance and immune modulation. The large‐scale processing of exosomes for use as therapeutic vectors has been proposed, but these applications are limited by impure, low‐yield recoveries from cell culture milieu (CCM). Current isolation methods are also limited by tedious and laborious workflows, especially toward an isolation of EVs from CCM for therapeutic applications. Employed is a rapid (<10 min) EV isolation method on a capillary‐channeled polymer fiber spin‐down tip format. EVs are isolated from the CCM of suspension‐adapted human embryonic kidney cells (HEK293), one of the candidate cell lines for commercial EV production. This batch solid‐phase extraction technique allows 10¹²EVs to be obtained from only 100‐µl aliquots of milieu, processed using a benchtop centrifuge. The tip‐isolated EVs were characterized using transmission electron microscopy, multi‐angle light scattering, absorbance quantification, an enzyme‐linked immunosorbent assay to tetraspanin marker proteins, and a protein purity assay. It is believed that the demonstrated approach has immediate relevance in research and analytical laboratories, with opportunities for production‐level scale‐up projected. 

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. 

Extracellular vesicles (EVs) are membrane-bound nanoparticles (50–1000 nm) secreted by all cell types and play critical roles in various biological processes. Among these, exosomes, a smaller subset of EVs, have attracted considerable interest due to their potential applications in diagnostics and therapeutics. However, conventional EV isolation methods are often limited by inefficiencies in processing time, recovery, and scalability. Hydrophobic interaction chromatography utilizing capillary-channeled polymer (C–CP) fiber stationary phases offers a promising alternative, enabling rapid (<15 min), cost-effective (~$5 per column) EV isolation with high loading capacities (~1010–10¹² particles) and minimal sample pre-processing. Despite these advantages, achieving high-throughput EV isolation for larger-scale applications using the C–CP fiber platform is the present challenge. To this end, further optimization of stationary phase packing and adsorption conditions is necessary to maximize the available binding surface area in the current microbore column format. This study systematically investigates the influence of interstitial fraction (i.e. packing density) in polyester (PET) C–CP fiber columns on the dynamic binding capacity (DBC) of EVs isolated from human urine using a high-performance liquid chromatography platform. Microbore columns (0.76 mm i.d. × 300 mm) packed with PET C–CP fibers in both an eight-channel (PET-8) and a novel trilobal (PET-Y) configuration were evaluated using breakthrough curves and frontal analysis. The results reveal that lower packing densities correlate with higher mass- and surface areabased EV binding capacities, with a maximum DBCs of 2.86 × 10¹³ EVs g-1 fiber and 1.22 × 10¹⁴ EVs m⁻² fiber achieved in <2 min of sample loading. Under optimum conditions, surface utilization of >50 % is realized. These results establish a framework for optimizing C–CP fiber-based platforms to enhance EV capture efficiency, facilitating the development of scalable EV isolation techniques for biomedical research and therapeutic applications. 

Cultured cell lines are very commonly used for the mass production of therapeutic proteins, such as monoclonal antibodies (mAbs). In particular, Chinese hamster ovary (CHO) cell lines are widely employed due to their high tolerance to variations in experimental conditions and their ability to grow in suspension or serum free media. CHO cell lines are known for their ability to produce high titers of biotherapeutic products such as immunoglobulin G (IgG). An emergent alternative means of treating diseases, such as cancer, is the use of gene therapies, wherein genetic cargo is “packaged” in nanosized vesicular structures, referred to as “vectors”. One particularly attractive vector option is extracellular vesicles (EVs), of which exosomes are of greatest interest. While exosomes can be harvested from virtually any human body fluid, bovine milk, or even plants, their production in cell cultures is an attractive commercial approach. In fact, the same CHO cell types employed for mAb production also produce exosomes as a natural byproduct. Here, we describe a single integrated 2D liquid chromatography (2DLC) method for the quantitative recovery of both exosomes and antibodies from a singular sample aliquot. At the heart of the method is the use of polyester capillary-channeled polymer (C−CP) fibers as the first dimension column, wherein exosomes/EVs are captured from the supernatant sample and subsequently determined by multiangle light scattering (MALS), while the mAbs are captured, eluted, and quantified using a protein A-modified C−CP fiber column in the second dimension, all in a 10 min workflow. These efforts demonstrate the versatility of the C−CP fiber phases with the capacity to harvest both forms of therapeutics from a single bioreactor, suggesting an appreciable potential impact in the field of biotherapeutics production. 

continuing emphasis. Polypropylene (PP) capillary-channeled polymer (C-CP) fiber columns are modified with the biotin- binding protein streptavidin (SAV) to capture biotinylated proteins. The loading characteristics of SAV on fiber supports were determined using breakthrough curves and frontal analysis. Based on adsorption data, a 3-min on-column loading at a flow rate of 0.5 mL min−1 (295.2 cm h−1) with a SAV feed concentration of 0.5 mg mL−1 produces a SAV loading capacity of 1.4 mg g−1 fiber. SAV has an incredibly high affinity for the small-molecule biotin (10−14 M), such that this binding relationship can be exploited by labeling a target protein with biotin via an Avi-tag. To evaluate the capture of the biotinylated proteins on the modified PP surface, the biotinylated versions of bovine serum albumin (b-BSA) and green fluorescent protein (b-GFP) were utilized as probe species. The loading buffer composition and flow rate were optimized towards protein capture. The non-ionic detergent Tween-20 was added to the deposition solutions to minimize non-specific binding. Values of 0.25–0.50% (v/v) Tween-20 in PBS exhibited better capture efficiency, while minimizing the non-specific binding for b-BSA and b-GFP, respectively. The C-CP fiber platform has the potential to provide a fast and low-cost method to capture targeted proteins for applications including protein purification or pull-down assays. 

Protein A (ProA) chromatography is a mainstay in the analytical and preparative scale isolation/purification of monoclonal antibodies (mAbs). One area of interest is continuous processing or continuous chromatography, where ProA chromatography is used in the large-scale purification of mAbs. However, filtration is required prior to all ProA isolations to remove large particulates in cell culture supernatant, consisting of a mixture of cell debris, host cell contaminants, media components, etc. Currently, in-line filters are used to remove particles in the supernatant, requiring replacement over time due to fouling; regardless of the scale. Here we demonstrate the ProA isolation of unfiltered Chinese hamster ovary (CHO) cell media using capillary-channel polymer (C-CP) fiber stationary phases modified with S. aureus Protein A (rSPA). The base polymer of the analytical scale C-CP columns costs ~$5 per 30 cm column, and when modified with ProA, the base cost is ~$25 per 30 cm column, a cost-effective option in comparison to analytical-scale commercial columns. To directly sample unfiltered media, a 5 cm gap was created at the head of the C-CP column, where the large particulates are trapped, while molecular solutes flow through the capillary channels without sacrifice in analytical performance, mAb loading capacity, or backpressure increases. The binding capacity of the gap ProA C-CP column was ~ 2 mg mL− 1 of IgG per bed volume. The same analytical column could be operated after processing a total of ~ 56 column bed volumes of supernatant (>25 analytical cycles) without the need for caustic clean-in-place processing.