Biomimetic Design Of Affinity Peptide Ligands For Human IgG And Its Affinity Chromatography

Monoclonal antibodies are of great application value in the treatment of inflammation, cancer, and infectious diseases. Among them, Immunoglobulin G(IgG) is one of the most important classes. The separation and purification of antibodis is the key to the whole production process. In comparison with other kinds of ligands, peptide ligands have several advantages. For example, they have higher structure stability, reduced ligand leaching, mild reaction condition, and they can in general better withstand the harsh cleaning in place(CIP) conditions such as 0.1 M NaOH. However by far, there are very few known peptide ligands. Therefore, research in designing high-af?nity peptide ligands is highly significant in the practical application of affinity chromatography.This thesis first established a biomimetic design strategy of af?nity peptide ligands based on IgG-(Protein A) SpA complex. The core of the strategy included four parts:(1) On the basis of the SpA-IgG complex, MM/PBSA free energy calculation coupled with free energy decomposition method was utilized to obtain 6 hot-spot residues of SpA in interaction with IgG, and thereby the SpA affinity binding motif was built;(2) Amino acid location method was used to determine the kind of inserted residues, and thus the peptide construction modes and a peptide ligand library were built;(3) Multiple molecular simulation screening methods were established, and thus a number of candidate peptide ligands were acquired;(4) Chromatographic experiments coupled with eletrophoresis analysis were used to verify high-affinity ligands. By far, three octapeptides, FYWHCLDE, FYCHWALE, and FYCHTIDE were confirmed to have high affinity interaction with IgG. Chromatographic analyses indicated that the affinity interactions between peptide ligands and IgG were dominated by electrostatic interactions. Kd values were in the range of 1.5-6.1 μM, and the binding capacities could reach 100-170 mg/m L. The octapeptide-based affinity chromatography was used to separate IgG from human serum sample in a single step, and the purified hIgG was obtained with a purity of up to 85-90%. MD simulations were used to analyze the affinity and binding strength within the three peptide-Fc complexes. It revealed that the binding occurred at the ―consensus binding site‖. At the same ligand density(10.4 μmol/m L), the binding capacity of hIgG was in the order of FYWHCLDE(104.2 mg/mL) > FYCHWALE(87.6 mg/m L) > FYCHTIDE(63.7 mg/mL).FYWHCLDE was top ranked in the peptide library. The effect of coupling ligand densities on adsorption equilibrium of h IgG was investigated. Four ligand densities were selected to study static adsorptions of hIgG and BSA. It was found that the affinity resin with a ligand density of 23.9 μmol/mL showed the highest selectivity to IgG. Breakthrough behavior of diluted serum proteins and hIgG indicated that dynamic binding capacity(DBC) increased with increasing ligand density. The affintiy resin was also applied to hIgG and mAb purifications from serum and cell culture medium, respectively. As a result, the eluted antibodies were obtained with high purities(95%) and recovery yields(89%).Next, any two of the three octapeptides were mixed and coupled to Sepharose gel, leading to the formation of three dual-ligand af?nity systems. Adsorption isotherms showed that there was synergistic effect of the two peptide ligands in a dual-ligand system, so the af?nity of a dual-ligand resin for h IgG was higher than those of its counterparts, single-ligand resins. Of the three dual-ligand systems, the FYWHCLDE–FYCHTIDE resin showed the highest af?nity. It was found that the synergistic effect increased with increasing the total ligand density of the two peptides in the dual-ligand af?nity system. Moreover, the FYWHCLDE–FYCHTIDE system at a molar ratio of 2:1 displayed the highest af?nity for h IgG(0.69 μM), indicating that the synergistic effect reached the maximum at this ratio.This thesis not only established a biomimetic design and screening strategy, but also has guiding significance for designing and screening of affinity peptide ligands for other important proteins. For example, the biomimetic design strategy was applied to build the affinity peptide library of erythropoietin(EPO). Firstly, on the basis of the three hot spot residues of EPObp1-EPO complex, a peptide library was established by the biomimetic design method. Then, same molecular simulation methods were utilized to screen the peptide library. Till now a total of 33 peptide candidates have been identified by the screening procedures prior to MD simulation. It is expected that further screening by MD simulation and experimental studies will produce high affinity peptide ligands for EPO.