Affinity Chromatography of IgG Based on Octapeptide Ligands Designed By Biomimetic Method

Staphylococcal Protein A (SpA) has been widely used as an affinity ligand for the purification of immunoglobulin G (IgG). However, SpA ligand is expensive and has a tendency to lose its activity when exposed to harsh washing and elution conditions. Moreover, SpA has a potential for inducing harmful immunogenic responses in patients upon likely leakage into antibody products. These issues have triggered a great interest in discovering and/or designing alternative affinity ligands for IgG. Herein, based on the affinity motif of SpA determiend from the IgG-SpA complex, we have developed a biomimetic design strategy for affinity peptide ligands of IgG. The method involves establishment of an affinity peptide model with the hot spots determined from the affinity motif of SpA, introduction of cysteine residue for ligand immobilization, and amino acid location to determine other amino acids for insertion. This led to the construction of a peptide library. The library was screened by using different molecular simulation protocols, resulting in the selection of 15 peptide candidates. Molecular dynamics simulations suggests that 14 of them would be high affinity ligands for IgG. The affinity and specificity of the top one ligand, FYWHCLDE, were exemplified by protein chromatography and IgG purification. The results indicate that the design strategy was successful and the affinity peptide ligand for IgG is promising for application in antibody purifications. Then, the effect of ligand density for IgG adsorption as well as purifications of human IgG and monoclonal antibody were studied. Finally, two more octapeptides, FYCHWALE and FYCHTIDE, were identified. Purifications of human IgG and monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. The molecular basis for the binding affinity of the peptides for the Fc fragment of IgG was elucidated by molecular dynamics simulations.