IgG-Binding\nPeptidomimetic Mixed-Charge Polymer-Modified\nResins for Chromatographic Purification of Antibodies

The process of antibody\npurification using Fc affinity ligands\nsuch as protein A, G, and L faces several challenges including high\ncost, low stability, and loss of antibody activity due to harsh elution\nconditions. Here, we describe a chromatographic purification of antibodies\nutilizing a pH-responsive mixed-charge polymer that mimics the IgG-binding\npeptide (Z34C) derived from the B domain of protein A. The protein\nA mimetic resins were prepared by modifying the surface of a TOYOPEARL,\nmethacrylate resin with a polymer that mimics the amino acid sequence\nof Z34C and the functions of histidine and acidic and neutral amino\nacids using histamine methacrylamide (HisMA), methacrylic acid, and\nneutral monomers. The therapeutic monoclonal antibody (mAb), rituximab,\nwas retained on the column at pH 7 and eluted under mildly acidic\nconditions at pH 5 using a protein A mimetic resin (HisMA20-EEMA)\noptimized for antibody interaction. The injected antibodies were selectively\ncaptured on the column by hydrophobic and electrostatic interactions\nwith the protein A mimetic polymer under neutral conditions and eluted\nby electrostatic repulsion under acidic conditions. The HisMA20-EEMA\ncolumn successfully purified mAbs from mixtures with BSA, mouse ascites\nfluid, and hybridoma cell culture supernatant. In addition, the HisMA20-EEMA\ncolumn consistently achieved 90% antibody recovery in 100 consecutive\npurifications from cell culture supernatant. The antibody purification\nmethod presented in this study is low cost, highly durable, easy to\nsynthesize, and allows for mild elution conditions. The results demonstrate\nthat the approach of mimicking IgG-binding peptides with mixed-charge\npolymers is useful for the development of column packing materials\nfor antibody purification.