FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody

Wei-Wei Zhao, Qing-Hong Shi, Yan Sun
Journal of Chromatography. A 2014 August 15, 1355: 107-14
This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles.


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