Cyclic peptide ligand with high binding capacity for affinitypurification of immunoglobulin G = Cyclic peptide ligand를이용한 IgG 정제

Abstract

The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has neces-sitated the development of antibody production and purification technologies for both academic andindustrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity andhave facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the presentstudy we describe the development of a reusable high affinity column for antibody purification utilizingimmobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys toSer substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins,packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Serone showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab).The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than wasrequired for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBPcolumns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swinehuman sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regeneratedand operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in thepresent study.