(336a) Novel Peptoid Ligands for IgG Purification

A great number of peptides with high affinity for protein targets is currently known and utilized as drugs, affinity ligands, and diagnostic agents. Recently, a broad spectrum of peptide mimetics has emerged, which hold considerable promise for replacing peptides in a variety of biotech and medical applications. Among them, peptoids in particular show excellent biorecognition properties, as well as higher chemical and biochemical stability as compared to peptides. This indicates the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with equal or higher affinity. To begin exploring this field, a small library of peptoid analogues of the IgG-binding peptide HWRGWV was constructed using both amino acid-like and non-natural functional groups, different arrangement of the residues, inclusion of spacing amino acids, and different length. Ligands with affinity for IgG were selected by screening the peptoid variants immobilized on solid supports via IgG binding tests in non competitive and, subsequently, competitive (cell culture supernatant added with bovine serum) conditions. The sequences offering the highest IgG recovery and purity were also tested to select those with the highest chemical and biochemical stability. To this end, the peptoids were challenged with a variety of chemical agents and proteolytic enzymes. Three peptoid-based adsorbents were finally characterized by binding isotherm to determine equilibrium capacity (Q_max) and strength of binding on solid phase (K_D,S). Selected peptoids were also modeled in silico to estimate the binding site on IgG and the binding strength in solution. To validate such predictions, one sequence was tested by HDX-MS and isothermal calorimetry, which confirmed both predicted binding site and true affinity (K_D,L) in solution. This study offers the first insight into the design of bioactive peptoids with equal or better affinity than their parental peptides, and an approach for overcoming the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.