title: Polyclonal and monoclonal IgG binding on protein A resins-Evidence of competitive binding effects.
abstract: Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, "shrinking core" behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. Biotechnol. Bioeng. 2017;114: 1803-1812. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213.
authors.forename: Justin
authors.identifier: 0000-0001-5325-6147
authors.initials: J
authors.lastname: Weinberg
authors.affiliation: Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904.
authors.forename: Shaojie
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213.
authors.forename: Gillian
authors.initials: G
authors.lastname: Crews
authors.affiliation: Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213.
authors.forename: Edward
authors.initials: E
authors.lastname: Healy
authors.affiliation: Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, Virginia 22904.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Carta
authors.affiliation: Department of Chemical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213.
authors.forename: Todd
authors.initials: T
authors.lastname: Przybycien
mesh_terms: D000327:Adsorption; D000837:Anion Exchange Resins / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D001665:Binding Sites; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D000837:Anion Exchange Resins; D061067:Antibodies, Monoclonal, Humanized; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A
keywords: Protein A chromatography; competitive binding; monoclonal antibodies; polyclonal antibodies