title: Chemical modifications in therapeutic protein aggregates generated under different stress conditions.
abstract: In this study, we characterized the chemical modifications in the monoclonal antibody (IgG(2)) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met(246), His(304), and His(427) in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxide-treated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.
authors.affiliation: Department of Analytical and Formulation Sciences, Amgen Inc., Thousand Oaks, California 91320, USA. qluo@amgen.com
authors.forename: Quanzhou
authors.initials: Q
authors.lastname: Luo
authors.forename: Marisa K
authors.initials: MK
authors.lastname: Joubert
authors.forename: Riki
authors.initials: R
authors.lastname: Stevenson
authors.forename: Randal R
authors.initials: RR
authors.lastname: Ketchem
authors.forename: Linda O
authors.initials: LO
authors.lastname: Narhi
authors.forename: Jette
authors.initials: J
authors.lastname: Wypych
mesh_terms: D000818:Animals; D003300:Copper / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000627:therapeutic use; D051379:Mice; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D007141:Immunoglobulin Fc Fragments; D003300:Copper; D006861:Hydrogen Peroxide