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.
Alleviation of the necessity for supernatant prefiltering in the protein a recovery of Monoclonal antibodies from Chinese hamster ovary cell cultures
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.
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- Award ID(s):
- 2107882
- PAR ID:
- 10535288
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Journal of Chromatography B
- Volume:
- 1230
- Issue:
- C
- ISSN:
- 1570-0232
- Page Range / eLocation ID:
- 123919
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
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.more » « less
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Extracellular vesicles (EVs) are 50–1000 nm membranous vesicles secreted from all cells that play important roles in many biological processes. Exosomes, a smaller-sized subset of EVs, have become of increasing interest in fundamental biochemistry and clinical fields due to their rich biological cargos and their roles in processes such as cell-signaling, maintaining homeostasis, and regulating cellular functions. To be implemented effectively in fundamental biochemistry and clinical diagnostics fields of study, and for their proposed use as vectors in gene therapies, there is a need for new methods for the isolation of large concentrations of high-purity exosomes from complex matrices in a timely manner. To address current limitations regarding recovery and purity, described here is a frontal throughput and recovery analysis of exosomes derived from human embryonic kidney (HEK) cell cultures and human urine specimens using capillary-channeled polymer (C-CP) fiber stationary phases via high performance liquid chromatography (HPLC). Using the C-CP fiber HPLC method for EV isolations, the challenge of recovering purified EVs from small sample volumes imparted by the traditional techniques was overcome while introducing significant benefits in processing, affordability (~5 $ per column), loading (~1012 particles), and recovery (1011–1012 particles) from whole specimens without further processing requirements.more » « less
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Abstract Extracellular vesicles, including microvesicles and exosomes, are lipidic membrane‐derived vesicles that are secreted by most cell types. Exosomes, one class of these vesicles that are 30–100 nm in diameter, hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. For exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Most current exosome isolation methods have practical problems including being too time‐consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary‐channeled polymer (C‐CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from diverse matrices of practical concern. Initial results demonstrate the ability to isolate extracellular vesicles enriched in exosomes with comparable yields and size distributions on a much faster time scale when compared to traditional isolation methods. As a demonstration of the potential analytical utility of the approach, extracellular vesicle recoveries from cell culture milieu and a mock urine matrix are presented. The potential for scalable separations covering submilliliter spin‐down columns to the preparative scale is anticipated.
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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.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.jchromb.2023.123919
