title: Protein A affinity precipitation of human immunoglobulin G.
abstract: The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit(®) S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1°C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes.
authors.affiliation: Bioseparation Engineering Group, Technische Universität München, 85748 Garching, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Janoschek
authors.affiliation: Bioseparation Engineering Group, Technische Universität München, 85748 Garching, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Freiherr von Roman
authors.affiliation: Bioseparation Engineering Group, Technische Universität München, 85748 Garching, Germany. Electronic address: s.berensmeier@tum.de.
authors.forename: Sonja
authors.initials: S
authors.lastname: Berensmeier
mesh_terms: D000180:Acrylic Resins; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000180:Acrylic Resins; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; C050528:Eudragit RS
keywords: Affinity precipitation; Antibody purification; Eudragit; Immunoglobulin G; protein A