title: Transient conformational modification of immunoglobulin G during purification by protein A affinity chromatography.
abstract: Exposure of three native IgG1 monoclonal antibodies to 100mM acetate, pH 3.5 had no significant effect on their hydrodynamic size (11.5±0.5nm), while elution from protein A with the same buffer created a conformation of 5.5±1.0nm. Formation of the reduced-size conformation was preceded by the known destabilization of the second constant domain of the heavy chain (Cγ2) by contact with protein A, then compounded by exposure to low pH, creating extended flexibility in the hinge-Cγ2 region and allowing the Fab region to fold over the Fc region. The reduced-size conformation was necessary for complete elution. It persisted unchanged for at least 7 days under elution conditions. Physiological conditions restored native size, and it was maintained on re-exposure to 100mM acetate, pH 3.5. Protein A-mediated destabilization and subsequent restoration of native size did not create aggregates, but the reduced-size conformation was more susceptible to aggregation by secondary stress than native antibody. Protein A-mediated formation of the reduced-size conformation is probably universal during purification of human IgG1 antibodies, and may occur with other subclasses and IgG from other species, as well as Fc-fusion proteins.
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore. Electronic address: pete_gagnon@bti.a-star.edu.sg.
authors.forename: Pete
authors.initials: P
authors.lastname: Gagnon
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore.
authors.forename: Rui
authors.initials: R
authors.lastname: Nian
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore.
authors.forename: Denise
authors.initials: D
authors.lastname: Leong
authors.affiliation: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore.
authors.forename: Aina
authors.initials: A
authors.lastname: Hoi
mesh_terms: D000085:Acetates / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002846:Chromatography, Affinity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification; D010315:Particle Accelerators; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000085:Acetates; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A
keywords: Arginine; Denaturation; IgG purification; IgG size; Protein A; Refolding