Octapeptide-based affinity chromatography of human immunoglobulin G: Comparisons of three different ligands

• Affinity chromatography of antibody based on three octapeptide ligands was reported.
• The ligands bound Fc (not Fab) and the binding sites were similar to those for SpA.
• Adsorption equilibria of hIgG were compared for the different affinity resins.
• hIgG and mAb purifications were achieved at high purities and recovery yields.
• MD simulations revealed molecular interactions of the octapeptides and Fc fragment.

In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)–Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0–6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64–104 mg/mL drained gel in the order of FYWHCLDE > FYCHWALE > FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations.