In-line coupling of capillary-channeled polymer fiber columns with optical absorbance and multi-angle light scattering detection for the isolation and characterization of exosomes.

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.

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures.

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

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 âˆ¼ 2 mg mL-1 of IgG per bed volume. The same analytical column could be operated after processing a total of âˆ¼ 56 column bed volumes of supernatant (>25 analytical cycles) without the need for caustic clean-in-place processing.

Quantitative Recoveries of Exosomes and Monoclonal Antibodies from Chinese Hamster Ovary Cell Cultures by Use of a Single, Integrated Two-Dimensional Liquid Chromatography Method.

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.

Two-dimensional separation of water-soluble polymers using size exclusion and reversed phase chromatography employing capillary-channeled polymer fiber columns.

Polymeric materials are readily available, durable materials that have piqued the interest of many diverse fields, ranging from biomedical engineering to construction. The physiochemical properties of a polymer dictate the behavior and function, where large polydispersity among polymer properties can lead to problems; however, current polymer analysis methods often only report results for one particular property. Two-dimensional liquid chromatography (2DLC) applications have become increasingly popular due to the ability to implement two chromatographic modalities in one platform, meaning the ability to simultaneously address multiple physiochemical aspects of a polymer sample, such as functional group content and molar mass. The work presented employs size exclusion chromatography (SEC) and reversed-phase (RP) chromatography, through two coupling strategies: SEC x RP and RP x RP separations of the water-soluble polymers poly(methacrylic acid) (PMA) and polystyrene sulfonic acid (PSSA). Capillary-channeled polymer (C-CP) fiber (polyester and polypropylene) stationary phases were used for the RP separations. Particularly attractive is the fact that they are easily implemented as the second dimension in 2DLC workflows due to their low backpressure (<1000 psi at ∼70 mm sec-1) and fast separation times. In-line multi-angle light scattering (MALS) was also implemented for molecular weight determinations of the polymer samples, with the molecular weight of PMA ranging from 5 × 104 to 2 × 105 g mol-1, while PSSA ranges from 105 to 108 g mol-1. While the orthogonal pairing of SEC x RP addresses polymer sizing and chemistry, this approach is limited by long separation times (80 min), the need for high solute concentrations (PMA = 1.79 mg mL-1 and PSSA = 0.175 mg mL-1 to yield comparable absorbance responses) due to on-column dilution and subsequently limited resolution in the RP separation space. With RP x RP couplings, separation times were significantly reduced (40 min), with lower sample concentrations (0.595 mg mL-1 of PMA and 0.05 mg mL-1 of PSSA) required. The combined RP strategy provided better overall distinction in the chemical distribution of the polymers, yielding 7 distict species versus 3 for the SEC x RP coupling.

Rapid metal speciation of cell culture media using reversed-phase separations and inductively coupled plasma optical emission spectrometry.

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.

Trace Elemental Analysis of Bulk Thorium Using an Automated Separation-Inductively Coupled Plasma Optical Emission Spectroscopy Methodology.

Presented here is a novel automated method for determining the trace element composition of bulk thorium by inductively coupled plasma-optical emission spectroscopy (ICP-OES). ICP-OES is a universal approach for measuring the trace elemental impurities present in actinide-rich materials; however, due to the emission rich spectrum of the actinide, a separation from the trace elements is warranted for spectrochemical analysis. Here, AG MP-1 ion exchange resin was utilized for retention of the Th matrix, while allowing the trace element impurities to be separated prior to subsequent analysis using ICP-OES. After demonstrating the separation on traditional gravity-driven columns, the methodology was transitioned to an automated platform for comparison. This automated platform utilizes syringe-driven sample and solvent flow and can collect the trace element and thorium fractions in separate locations. While reducing the sample size (500 µL, 1.5 mg of Th), maintaining the overall separation efficiency (recoveries >95%), and illustrating the sample throughput ability (n = 10+), this automated methodology could be readily adopted to nuclear facilities in which the determination of trace elemental impurities in Th samples is warranted.