Protein A (ProA) chromatography is a bioseparations technique employed throughout the biopharmaceutical industry for the selective capture and purification of IgG-class monoclonal antibodies (mAbs) and Fc-fusion proteins. The rapid growth of mAbs as commercial therapeutics has motivated the need for improved, efficient, and high-throughput purification processes during manufacturing. In direct response, the work presented in thesis aims to 1) increase the scientific community’s understanding of IgG adsorption behavior on ProA chromatography resins and 2) improve the performance of ProA chromatography with ligands that are chemically modified using polyethylene glycol (PEGylated). The results of this thesis suggest that IgG molecules of varying binding strength, or varying elution pH, are capable of competing for binding sites on ProA chromatography resins in simultaneous or sequential adsorption. The competitive phenomenon derives from variance in IgG binding strength, or IgG elution pH, due to differences in sub-class behavior as well as secondary IgG binding interactions with the ProA ligand. Competition is readily apparent in the adsorption of human polyclonal IgG, which has a wide variety of IgG sub-classes and binding epitopes. Additionally, the results presented in this thesis suggest that ProA chromatography resins with PEGylated ligands are a viable path to increase resin robustness and real-world chromatographic selectivity. It is demonstrated that ligand PEGylation can increase resistance to proteolytic digestion, mitigate impurity interactions with mAbs that are bound to ProA, and increase process selectivity against Chinese Hamster Ovary host cell proteins by up to 37%. However, resins with large volumes of conjugated PEG significantly decrease IgG static binding capacity and decrease the available pore space for diffusion, resulting in losses in dynamic binding capacity and productivity. Lighter modifications appear to avoid losses in dynamic binding capacity, however, they do not appear to be effective at mitigating impurity interactions with mAbs that are bound to ProA, which is key to increasing process selectivity. PEGylation of ProA also universally increases the elution pH of IgG molecules by weakening the binding interaction. This last result opens another path of viability for PEGylated ProA ligands for purification of mAbs of Fc-fusion proteins that are sensitive to low pH environments.
| Identifer | oai:union.ndltd.org:cmu.edu/oai:repository.cmu.edu:dissertations-2114 |
| Date | 01 December 2017 |
| Creators | Weinberg, Justin B. |
| Publisher | Research Showcase @ CMU |
| Source Sets | Carnegie Mellon University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations |
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