title: Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column. Part I. Chromatographic elution and batch adsorption behavior.
abstract: A glycosylated IgG2 monoclonal antibody exhibits a two-peak elution behavior when loaded on a strong cation exchange column and eluted with either a linear salt gradient or two salt steps at increasing salt concentrations. The two-peak behavior is more pronounced for conditions where the initial antibody binding is stronger, i.e. at lower pH and buffer concentration, where the hold time prior to elution is longer, where the protein mass load is lower, and where the load flow rate is higher. The effect is also dependent on the resin type, being prominent for the polymer-functionalized resin Fractogel EMD SO₃(-) and virtually absent for a macroporous resin with similar backbone but no grafted polymers. Size exclusion chromatography and dynamic light scattering show that the early eluting peak consists exclusively of the native monomeric species while the late eluting peak is a mixture of monomeric and aggregated species. Batch adsorption/desorption experiments show that the bound protein can be desorbed in two steps, with a fraction desorbed in 0.33 M NaCl, corresponding to native monomer, and a second fraction desorbed in 1M NaCl. The latter fraction decreases with protein mass load and becomes almost negligible when the resin is initially completely saturated with protein. Confocal laser scanning microscopy showed that the two-peak elution/desorption behavior is related to the unique kinetics of protein binding in the Fractogel resin. Following partial loading of the resin, the bound protein migrates toward the center of the particles during a hold step and is redistributed across the particle volume attaining low local bound protein concentrations. For these conditions the protein is apparently destabilized forming a strongly-bound unfolded intermediate that, in turn, generates aggregates upon elution in high salt.
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Shaojie
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA. Electronic address: gc@virginia.edu.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Carta
mesh_terms: D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D002411:Cation Exchange Resins / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods; D004553:Electric Conductivity; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification; D018613:Microscopy, Confocal; D008970:Molecular Weight; D011485:Protein Binding; D055503:Protein Multimerization; D058767:Protein Unfolding
chemical_list: D000911:Antibodies, Monoclonal; D002411:Cation Exchange Resins; D006023:Glycoproteins; D007074:Immunoglobulin G
keywords: Aggregation; Cation exchange; Monoclonal antibody; Unfolding