Oriented Covalent Immobilization of Engineered ZZ-Cys onto Maleimide-Sepharose: An Affinity Platform for IgG Purification

Abstract

Protein A affinity chromatography is an important technique that is widely used in purifying polyclonal and monoclonal antibodies. However, improving the IgG loading capacity of protein A affinity materials remains crucial. In this study, a smaller divalent IgG binding molecule derived from the B domain of protein A, i.e., ZZ-domain, was used to develop an affinity adsorbent with high IgG loading capacity by improving the unit area yield of the site-specific immobilization affinity ligand. The engineered ZZ-Cys was tightly immobilized onto Sepharose support via the covalent incorporation of a cysteine handle and a maleimide group, with oriented manner and divalent IgG binding capacity, thereby resulting in homogenous conjugates, namely, Sepharose–ZZ^SA. Approximately 1.19 mg of ZZ-Cys was coupled onto wet Sepharose g⁻¹ and the maximum saturation binding capacity of Sepharose–ZZ^SA g⁻¹ was approximately 23.80 mg of IgG. The smaller engineered ZZ-Cys can be produced at a lower cost than protein A and covalently conjugated onto matrix surface with high density and full IgG binding capacity. Thus, the proposed platform may be of general use for IgG purification in an efficient and economical manner.