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.

Neutron reflectivity measurement of protein A–antibody complex at the solid-liquid interface

“…Estimates of rSPA surface coverage were compared to approximations of protein A coverage on the porous glass resin Prosep (Millipore); it was inferred that surface coverage estimates for rSPA were within the expected range, and towards the low end. Apparently low protein A surface coverage coupled with IgG elution at pH 4.1 suggested that each IgG was bound to only one protein A, supporting a “single/dual site binding” theory described by Gagnon and Nian [3]. Analysis of the reflectivity data revealed layer-based model structures in which it was possible to identify distinct contributions from protein A and from IgG4.…”

“…This elution pH is slightly higher than would typically be expected for elution of IgG from protein A [39], [40], [41], [42]. However, recent work by Gagnon and Nian has shown that IgG may interact with protein A by a dual-site binding mechanism in typical chromatography column environments [3], with “single site bound” IgG populations eluting at a higher pH. Thus, it is possible that the packing density of rSPA crosslinked to the surface was such that each IgG4 molecule bound a single protein A ligand, resulting in near-total elution at pH 4.1.…”

“…Therefore, despite uncertainty regarding interpretation of the model, the model itself can be considered an accurate representation of the interfacial macro-structure for the given system. A related consideration is that IgG has been shown to bind two protein A ligands simultaneously [3]. Due to the relatively sparse surface coverage of protein A, we suggest that this was not the case.…”

“…Due to the relatively sparse surface coverage of protein A, we suggest that this was not the case. Further, near-complete elution of the IgG at pH 4.1 suggests that IgG molecules were “single site bound” [3].…”

“…In two related studies, it was shown that IgG1 undergoes significant changes in average hydrodynamic radius and secondary structure content during elution in protein A chromatography [2], [3]. Gagnon and Nian [3] suggested that IgG1 size-reduction was influenced largely by protein concentration, a theory supported by other studies [4]; changes in secondary structure were proposed to be the result of a “dual site” interaction of IgG with protein A, whereby each IgG molecule bound two protein A molecules [3].…”