Membrane adsorbers and novel affinity peptides for recombinant protein purification
Date
2015
Authors
Weaver, Justin, author
Wickramasinghne, S. Ranil, advisor
Qian, Xianghong, committee member
Carlson, Jon O., committee member
Bailey, Travis S., committee member
Journal Title
Journal ISSN
Volume Title
Abstract
The purification of recombinant proteins for use as pharmaceutically active ingredients represents one of the largest costs of drug development and production. Of the different classes of recombinant protein therapeutics monoclonal antibodies represent the largest percentage of protein therapeutics currently on the market with even more in clinical development. The work presented in this thesis describes the evaluation of both commercial and newly designed anion exchange and hydrophobic interaction (HIC) membrane adsorbers as well as identification of novel affinity peptides for the purification of recombinant proteins, specifically monoclonal antibodies. Commercially available anion-exchange membrane adsorbers were evaluated for their potential to remove impurities commonly present at low concentration in recombinant protein solutions expressed in mammalian cell culture. These so-called trace impurities include virus, host cell proteins, and DNA; these impurities are of particular concern because they are highly immunogenic at very low concentrations. Ionic strength and pH were shown to be the dominant factors affecting impurity binding on quaternary amine (Q) membranes indicating these ligands interact with the impurities primarily through electrostatic interactions. It is likely impurity interactions with primary amine ligands involved not only electrostatic but hydrogen bonding interactions which stabilized impurity-ligand interactions enabling greater removal at a broader range of solution pH and ionic strength conditions. Binding of host cell proteins with a broad range of isoelectric points was also demonstrated using the primary amine ligand as compared to the Q ligands. The effect of solution pH, ionic strength, flow rate, and the presence of competing anionic species was investigated. In addition to commercially available anion-exchange membrane adsorbers novel anion-exchange membranes, developed by Dr. Bharat Bhut and Prof. Scott Husson at Clemson University, were evaluated for binding capacity and virus removal. Regenerated cellulose microfiltration membranes were modified with a negatively-charged quaternary amine polymer, systematically varying the polymer chain density and length. IgG and DNA binding capacity, as well as minute virus of mice removal, was evaluated as a function of polymer chain density and length. It was shown that IgG binding capacity increased with polymer chain density indicating IgG access to binding sites was not a limiting factor. Similarly, high polymer chain density and longer polymerization time (translating to longer polymer chain length) resulted in higher DNA binding and virus removal again indicating ligand accessibility was not an issue even with large solutes such as virus. Environmentally-responsive hydrophobic interaction membranes were also developed in the Wickramasinghe lab and evaluated for protein binding capacity and recovery. Three-dimensional polymer brushes were grafted from 0.45 µm pore size regenerated cellulose membrane surfaces. The dynamic binding capacity of human IgG was greater than current commercially available hydrophobic interaction membranes with comparable recoveries. Affinity purification using novel small peptides was also explored as an antibody purification tool. Several heptapeptide affinity ligands were identified that bound specifically to the Fc region of IgG. These peptides have similar function to Staphylococcus Aureus Protein A, which is used extensively as an affinity purification ligand for monoclonal antibodies in the pharmaceutical industry. A large library of seven amino acid-long peptides was screened via M13 Phage Display for specific binding to the Fc, or constant region, of human IgG antibody. After initial identification, specificity of binding only to IgG was demonstrated through subsequent competitive ELISA assays. Though the affinity peptides were initially screened against human IgG₄ Fc, binding to a larger subset of human and non-human antibodies was shown indicating the peptides were binding to highly conserved regions on the antibodies. Because Protein A has some limitations in industrial process applications, these novel heptapeptides may provide an alternative solution for affinity purification of monoclonal antibodies.
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Subject
antibody
peptides
adosrber
protein a
membrane