(471e) Equilibrium and Dynamics of Human IgG Adsorption to a Peptide Affinity Ligand On Pure and Mixed Monolayers

In this study, the behavior of a hexameric affinity peptide was characterized in depth and its potential as a means of immunoglobulin G (IgG) detection was explored. Surface Plasmon Resonance (SPR) was used to study the binding of human IgG to the hexameric peptide affinity ligand HWRGWV, which is covalently grafted to pure or mixed alkanethiol self-assembled monolayers (SAM) on gold surfaces. The reactive alkanethiol (HS-(CH₂)₁₁-(CH₂CH₂O)₆-NH₂, shortened notation, EG₆NH₂) is -NH₂ terminated, contains six ethylene glycol units, and was used to create pure monolayers on gold sensor surfaces. The NH₂ termini are reactive towards the C-terminus of the peptide ligands. To prepare mixed monolayers with the above thiol, a ‘dilutor’ thiol (HS-(CH₂)₁₁-(CH₂CH₂O)₃-OH, shortened notation, EG₃OH) with three ethylene glycol units and a hydroxyl terminus group, was utilized. The thiols were mixed at various percentages of EG₆NH₂ (with 0% denoting pure EG₃OH, 100% denoting pure EG₆NH₂) to form monolayers of varying NH₂densities on gold sensors and slides.

The pure or mixed monolayer surface density and peptide grafting density were quantified using Time of Flight Secondary Ion Mass Spectrometry (ToF SIMS) and ellipsometric measurements. The absence of phase islands of mixed monolayers was verified with contact angle measurements and the successful grafting of the peptide on these monolayers was also verified through ToF-SIMS. Peptides were covalently grafted via their C-termini to produce active SPR sensors and various protein binding experiments were carried out with SPR. The dynamics and extent of peptide-IgG binding were compared with results from conventional systems based on Protein A attached on gold via disulfide monolayers. Equilibrium and kinetic binding parameters for IgG were obtained and compared, for peptide ligands grafted on to pure and mixed monolayer systems, in order to make a comprehensive study of the roles of N-terminal capping, of peptide density and of the base matrix components on the IgG-peptide binding. Non specific binding was investigated by BSA binding experiments. The behavior of complex mixtures (Chinese Hamster Ovary supernatant, or CHO, containing IgG and other impurities) on the peptide sensors was studied, and was compared with pure IgG binding behavior. Different regeneration buffers were tested to obtain the optimum buffer, providing maximum regeneration, without affecting the peptide performance. The grafting density and the fractional conversion of NH₂ moeities of the monolayer to peptides, do not seem to play a significant role in the capacity or affinity of IgG binding, but the ratio of active thiol EG₆NH₂ to dilutor thiol EG₃OH has an effect on the selectivity of binding.  The optimum selectivity of IgG to BSA binding was obtained with peptides attached to mixed monolayers formed from 10% EG₆NH₂-90% EG₃OH thiol solution in ethanol.  IgG binding to acetylated HWRGWV (Ac-HWRGWV) supported on 100% EG₆NH₂ SAM showed an equilibrium dissociation constant, K_d of 5.8x 10^-7 M and that for HWRGWV on the 100% EG₆NH₂ SAM (after deprotection of Fmoc-HWRGWVA) yielded K_d of 1.2x10^-6 M. On the peptide with mixed monolayer system the K_d was 9.33x10^-7 M. Maximum binding capacities, Q_m were 3.8, 4.2 and 3.2 mg m^-2 for 100% EG₆NH₂-Ac-HWRGWV, 100% EG₆NH₂-HWRGWV and 10% EG₆NH₂-Ac-HWRGWV respectively. Real-time SPR data were analyzed with mass transport effects taken into consideration and were used to determine the apparent rate constants for association and all three peptide based systems yielded similar values for k_a (2.2, 2.1 and 1.9 m³ mol^-1 s^-1, respectively).  In binding experiments for IgG from complex mixtures, it was observed that that for diluter mixtures, the mixed monolayer system showed better performance, while for complex mixtures with higher IgG content, the binding results with pure monolayer + peptides conformed better with that of pure IgG mixture solutions. The regeneration-binding cycles for 100% EG₆NH₂-Ac-HWRGWV as well as 10% EG₆NH₂- Ac-HWRGWV systems showed optimum performance with 10% acetonitrile mixed with 0.1 N NaOH.  At least 8 cycles of regeneration and binding were demonstrated with little or no change in performance of the peptides, both on pure and mixed monolayers, with this regeneration buffer. The experiments carried out in the study helped understand the mechanism of binding of IgG to the small molecule ligands. These and previous experimental results by Yang et al working with HWRGWV peptide^{1, 2}indicate that neither hydrophobic nor electrostatic interactions are a dominant force in binding and that hydrogen bonding between residues play a major role in the specific affinity binding. The results of regeneration experiments carried out in this study indicate that merely the change of pH is not enough to cause 100% breaking of bonds between IgG and the peptide, as seems to be the case for Protein A regeneration. An introduction of a chaotropic agent (eg urea) or a polar solvent (eg. acetonitrile) is required to weaken forces contributed by hydrogen bonding. Results of this study can help formulate an effective biosensor for hIgG, and also helps further understand the mechanism of binding between hIgG and small molecule affinity ligand HWRGWV.

1.    Yang, H. O.; Gurgel, P. V.; Williams, D. K.; Bobay, B. G.; Cavanagh, J.; Muddiman, D. C.; Carbonell, R. G., Binding site on human immunoglobulin G for the affinity ligand HWRGWV. Journal of Molecular Recognition 2010,23, (3), 271-282.

2.    Yang, H.; Gurgel, P. V.; Carbonell, R. G., Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography. Journal of Chromatography A 2009, 1216, (6), 910-918.