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

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.

Figure 1.

Diagrammatic representation of the 2DLC coupling of the C–CP fiber HIC and ProA columns for the isolation of exosomes and IgG from CHO cell culture supernatants. (a) Fluid path for the initial sample introduction and (b) valving for the elution of the respective column isolates.

Figure 2.

Demonstrative chromatographs for the individual isolation and recovery processes employed for the isolation of therapeutics from CHO cell culture supernatants. (a) Chromatogram of 3-step HIC isolation of exosomes. The sample is introduced in 2 M ammonium sulfate, where salts and sugars are unretained. Proteins are eluted around 6 min with 1 M ammonium sulfate and 25% ACN in 1X PBS, and exosomes are eluted around 12 min with 40% ACN in 1X PBS. (b) Chromatogram of ProA isolation of IgG, with sample injected in 50 mM sodium phosphate, pH 7.4, and IgG elution with 150 mM citric acid, pH 2.6. In each case, a 20 μL supernatant aliquot is injected.

Figure 3.

(a) Chromatogram of the elution of the protein fraction of CHO cell supernatant from the PET C–CP fiber column under HIC conditions. Injection conditions: 2 M ammonium sulfate; elution conditions: 1 M ammonium sulfate +25% ACN. The entire fraction of protein elution is comprehensively transferred to ²D. (b) Chromatogram obtained at the exit of the ²D ProA C–CP fiber column. Comprehensive transfer with an 80 μL injection loop at intervals of 0.5 min. Injection mobile phase 50 mM sodium phosphate, pH 7.4 from 0–23 min. Elution of captured IgG initiated at 26 min with 150 mM citric acid, pH 2.6. The orange-shaded portions in each chromatogram represent the transfer fraction from ¹D to ²D.

Figure 4.

Product chromatograms for the separate stages of the isolation of a) exosomes and b) IgG from a 20 μL injection of CHO cell culture supernatant. (a) ¹D HIC isolation of exosomes with absorbance detection. TEM image of a single exosome (magnification = ×100.0K, acc. voltage = 120.0 kV). (b) ²D ProA isolation of IgG with column placed in the switch valve loop and absorbance detection. The orange region of each chromatogram represents the time frame where the two columns are in-line with each other.

Figure 5.

Multiangle light scattering (MALS) sizing of CHO cell-supernatant-derived EVs following HIC separation. Diameters of EVs ranged from 15–395 nm with a mean of 157 nm. Silica bead standard exhibits diameters ranging from 239–247 nm, in good agreement with known values.