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Free, publicly-accessible full text available March 1, 2025
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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.more » « lessFree, publicly-accessible full text available December 5, 2024
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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.more » « less
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Abstract 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.