Kinetic Analysis of the Multivalent Ligand Binding Interaction between Protein A/G and IgG: A Standard System Setting

Abstract

Recombinant protein A/G (PAG) has a sequence coding for eight IgG binding sites and has enhanced interspecies affinity. High-frequency sampling of a PAG titration with IgG produces concentration profiles that are sensitive to the kinetic availability of the binding sites. The full kinetic model developed here for IgG binding sequentially to PAG shows only two distinct kinetic processes, describing an initial rapid association of two antibodies to PAG with a rate constant k-fast = (1.86 ± 0.08) × 10 M s and a slower antibody binding process to all remaining sites, k-slow = (1.24 ± 0.05) × 10 M s. At equilibrium (after 1 h), the maximum IgG occupancy of PAG is 2.8 ± 0.5, conflicting with the genetic evidence of eight binding sites and suggesting significant steric hindrance of the neighboring IgG binding sites. The phosphate-buffered saline (PBS) solution defines a standard system setting, and this may be compared with other settings. The mean association rate of PAG-IgG in the standard setting is 282 ± 20% higher than when PAG is tethered to a surface. A systems biology approach requires that a model parameter set that defines a system in a standard setting should be transferable to another system. The transfer of parameters between settings may be performed using activity coefficients characterizing an effective concentration of species in a system, a = γc. The activity correction, γ, for the eight-site occupancy is γ = 0.35 ± 0.06, and mapping from the standard setting to the solution setting suggests γ = 0.4 ± 0.03. The role of activity coefficients and transferability of kinetic parameters between system settings is discussed.

Mechanisms of noncanonical binding dynamics in multivalent protein–protein interactions

“…Despite these challenges, the numerous applications of multivalency have led to many iterations of experimental and theoretical model systems designed to describe, predict, and explore particular aspects of these interactions (28)(29)(30). However, because the biophysical features are unique to each interaction pair, multivalent models are often tailored in a system-specific manner (31,32), having an in-depth focus on a subset of the parameter space (33)(34)(35) and using fitted parameters to account for nonideal behaviors and unmeasurable properties (36,37). For example, many descriptions have focused on the important role of valency in the binding mechanisms of complexes such as dimeric zincfinger transcription factors and bivalent IgG antibodies (30).…”

A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material

“…The binding kinetics and monomeric purity of 8 antibody samples was assessed using our novel biophotonic multiplexed biosensor platform, described in detail elsewhere [19, 20, 22]. Briefly, gold nanoparticles are printed onto an aminated glass surface using an inkjet printer and the nanoparticles are chemically grown on the surface to ~60 nm in diameter.…”

“…The Fc assay is calibrated on each array by injecting the NISTmAb at concentrations of 1.56, 3.23, and 6.25 nM and the response of the Fc and Fab assays recorded simultaneously. The data were used for kinetic analysis of the NISTmAb with a global fit to the Langmuir adsorption isotherm [19, 20]. The arrays were regenerated with a 0.01 M solution of phosphoric acid for 100 s between samples.…”

“…In this paper, we report a novel multiplex technique to simultaneously assess antibody affinity and monomeric purity, based on our in-house biosensor platform [19, 20]. Sensor surfaces are functionalised with either antigen or the Fc binding protein A/G (PAG) to generate assays for Fab and Fc fragments.…”

“…Sensor surfaces are functionalised with either antigen or the Fc binding protein A/G (PAG) to generate assays for Fab and Fc fragments. PAG has 3 sterically available binding sites for the Fc region of intact IgG and binds with high affinity to antibodies from several species [20]. The technique described here is comprised of two steps, shown in Fig.…”