title: Partial unfolding of a monoclonal antibody: role of a single domain in driving protein aggregation.
abstract: We have examined the effect of incubating a monoclonal antibody (mAb) in low (0-2.0 M) concentrations of guanidine hydrochloride (GdnHCl) on the protein's conformation and aggregation during isothermal incubation. In GdnHCl solutions at concentrations from 1.2 to 1.6 M, the mAb was partially unfolded. As demonstrated by fluorescence and circular dichroism spectroscopy, the partially unfolded state of the antibody had perturbed tertiary structure but retained native secondary structure. Furthermore, partial unfolding of the antibody was documented by analytical ultracentrifugation, dynamic light scattering, and limited proteolysis. Subsequent aggregation of the antibody was characterized using size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering. Over the entire concentration range (0-2.0 M) of GdnHCl, protein-protein interactions were attractive, as quantified by negative osmotic second virial coefficients measured with static light scattering. However, during isothermal incubation at 37 °C, the aggregation of the antibody was detected only in solutions that induced partial unfolding. Differential scanning calorimetry studies showed that the antibody's CH2 domains were unfolded in antibody molecules that had been incubated in 1.2 M and higher concentrations of GdnHCl. These results suggest that unfolding of the CH2 domains leads to aggregation.
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
authors.forename: Shyam B
authors.initials: SB
authors.lastname: Mehta
authors.forename: Jared S
authors.initials: JS
authors.lastname: Bee
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000502:physiology; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D019791:Guanidine