Z_Ca: A Protein A-Derived Domain with Calcium-Dependent Affinity for Mild Antibody Purification

Therapeutic antibodies are at the forefront of modern medicine where high purity, which is typically obtained by Protein A-based affinity purification, is of utmost importance. In this chapter, we present a method for neutral and selective purification of antibodies by utilizing an engineered affinity ligand, Z_Ca, derived from Protein A. This domain displays a calcium-dependent binding of antibodies and has been multimerized and immobilized to a chromatography resin to achieve an affinity matrix with high binding capacity. IgG antibodies can be eluted from the tetrameric Z_Ca ligand at pH 7 with the addition of EDTA, or at pH 5.5 with EDTA for purification of monoclonal IgG1, which is significantly milder than the low pH (3-4) required in conventional Protein A affinity chromatography. Here, a protocol for selective capture of IgG with elution at neutral pH from a Z_Ca tetramer ligand immobilized on a chromatography resin is described.