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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. 

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. 

There is great interest in advancing methodologies for the isolation and characterization of exosomes (30–150 nm, extracellular vesicles (EVs)) for fundamental biochemical research and liquid biopsy applications. This is due to the accessibility of exosomal surface biomarkers, providing relevant biochemical information from their cells of origin. Exosome-based techniques hold potential for diagnostic applications through less invasive sampling ( versus the physical extraction methods of pathology). This study demonstrates a simple spin-down tip methodology for generic exosome capture, followed by immunoaffinity-based fluorescent labeling to classify EVs captured on a polyester capillary-channeled polymer (C-CP) fiber stationary phase. An antibody to the generic EV tetraspanin protein (CD81) is employed to confirm the presence of biologically active EVs on the fiber surface. An antibody to the CA125 protein, upregulated in the case of ovarian cell stress, is included as a cancer marker protein. Scanning electron microscopy and confocal fluorescence microscopy were performed directly on the capture fibers to visualize the morphology and assess the bioactivity/identity of captured vesicles. This report provides a proof-of-concept for an efficient means of isolating, purifying, immunolabeling, and fluorescent imaging for the biomarker assessment of extracellular vesicles on a single platform . Herein lies the novelty of the overall approach. The ability to affect the entire isolation, immunolabeling, and imaging process in <5 hours is demonstrated. The C-CP fiber spin-down tip is an efficient exosome isolation methodology for microliter samples from diverse media (human urine and cell culture media here) towards diverse means of characterization and identification. 

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.

 

Cell culture media metal content is critical in mammalian cell growth and monoclonal antibody productivity. The variability in metal concentrations has multiple sources of origin. As such, there is a need to analyze media before, during, and after production. Furthermore, it is not the simple presence of a given metal that can impact processes, but also their chemical form that is, speciation. To a first approximation, it is instructive to simply and quickly ascertain if the metals exist as inorganic (free metal) ions or are part of an organometallic complex (ligated). Here we present a simple workflow involving the capture of ligated metals on a fiber stationary phase with passage of the free ions to an inductively coupled plasma optical emission spectrometry for quantification; the captured species are subsequently eluted for quantification. This first level of speciation (free vs. ligated) can be informative towards sources of contaminant metal species and means to assess bioreactor processes.

 

Capillary‐channeled polymer (C‐CP) fibers are demonstrated as a selective stationary phase for phosphopeptide analysis via LC–MS. Taking advantage of the oxidative self‐polymerization of dopamine under alkaline conditions, a simple system involving a dilute aqueous solution of 0.2% w/v dopamine hydrochloride in 0.15% w/v TRIS buffer, pH 8.5 was utilized to coat polydopamine onto nylon 6 C‐CP fibers. Confirmation of the polydopamine coating on the fibers (nylon‐PDA) was made through attenuated total reflection‐FTIR (ATR‐FTIR) analysis. Imaging using SEM was also performed to examine the morphology and topography of the nylon‐PDA. Subsequent loading of Fe³⁺to the nylon‐PDA matrix was confirmed by SEM/energy dispersive X‐ray spectroscopy (SEM/EDX). The Fe³⁺‐bound nylon‐PDA fibers packed in a microbore column format were tested in the off‐line preconcentration of phosphopeptides from a 1:100 mixture of β‐casein/BSA digests for MALDI‐TOF analysis. The packed column was also installed onto an HPLC system as a platform for the online sample clean‐up and enrichment of phosphopeptides from a 1:1000 mixture of β‐casein/BSA protein digests that were determined by subsequent ESI–MS analysis.