title: Nonnative aggregation of an IgG1 antibody in acidic conditions: part 1. Unfolding, colloidal interactions, and formation of high-molecular-weight aggregates.
abstract: Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5-5.5). A combination of intrinsic/extrinsic fluorescence (FL), circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed nonnative secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally and chemically induced aggregates showed similar ThT binding and secondary structural changes, and were noncovalent based on dissociation in concentrated guanidine hydrochloride solutions. Changes in intrinsic FL during chemical versus thermal unfolding suggest a greater degree of structural change during chemical unfolding, although aggregation proceeded through partially unfolded monomers in both cases.
authors.affiliation: Department of Chemical Engineering and Center for Molecular and Engineering Thermodynamics, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Rebecca K
authors.initials: RK
authors.lastname: Brummitt
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Nesta
authors.forename: Liuquan
authors.initials: L
authors.lastname: Chang
authors.forename: Susan F
authors.initials: SF
authors.lastname: Chase
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Laue
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Roberts
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D052160:Benzothiazoles; D001702:Biopharmaceutics / Q000379:methods*; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003102:Colloids / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D008027:Light; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D058767:Protein Unfolding; D012542:Scattering, Radiation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D052160:Benzothiazoles; D003102:Colloids; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D013844:Thiazoles; C009462:thioflavin T