title: Conformational plasticity of IgG during protein A affinity chromatography.
abstract: Single step elution of a protein A column with 100mM acetate pH 3.5 produced a curvilinear gradient with pH dropping steeply at first then more gradually as it approached endpoint. IgG with a native hydrodynamic diameter of 11.5 nm began to elute at pH 6.0 with a size of 9.4 nm. IgG size continued to decrease across the peak, reaching a minimum of 2.2 nm at pH 3.9. Secondary structure of early eluting IgG was only mildly affected but later eluting fractions became increasingly non-native with the 2.2 nm population exhibiting the highest proportion of β-sheet and lowest random coil of all conformations. Size reduction and structural change of IgG through this portion of the elution peak were attributed dominantly to a pre-existing tendency of highly concentrated IgG to adopt reduced size conformations at low pH and conductivity, facilitated by the known conformational relaxation of IgG by its interaction with protein A. IgG size increased to 10.4 nm as elution pH approached 3.5 across the tailing fractions. Major loss of β-sheet and increase of α-helix and random coil were observed in parallel. Late elution of this population was attributed to it being eluted from interactions with 2 distinct protein A domains, one bound to each side of the Fc region, creating a higher dissociation constant than single-site Fc-protein A interactions, and requiring more severely disruptive conditions for elution. The high degree of conformational disruption was attributed to simultaneous interaction of both heavy chains with protein A.
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore. Electronic address: pete_gagnon@bti.a-star.edu.sg.
authors.forename: Pete
authors.initials: P
authors.lastname: Gagnon
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, 138668, Singapore.
authors.forename: Rui
authors.initials: R
authors.lastname: Nian
mesh_terms: D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013205:Staphylococcal Protein A / Q000032:analysis*
chemical_list: D007074:Immunoglobulin G; D013205:Staphylococcal Protein A
keywords: Conformational modification; Elution; IgG concentration; IgG size; Protein A