Site-Specific Hydrolysis Reaction C‑Terminal of Methionine in Met-His during Metal-Catalyzed Oxidation of IgG‑1

• Published in: Molecular pharmaceutics Vol. 13; no. 4; pp. 1317 - 1328
• Main Authors: Mozziconacci, Olivier, Arora, Jayant, Toth, Ronald T, Joshi, Sangeeta B, Zhou, Shuxia, Volkin, David B, Schöneich, Christian
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04-04-2016
• Subjects: Catalysis; Chromatography, Gel; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Immunoglobulin G - chemistry; Metals - chemistry; Methionine - chemistry; Oxidation-Reduction; Spectrometry, Fluorescence; immunoglobulin; methionine; Fenton oxidation; IgG; sulfide radical cation; hydrolysis; histidine
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/acs.molpharmaceut.5b00944

The metal-catalyzed oxidation by [FeII(EDTA)]2–/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1–34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739–755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3–6%) were, however, observed in the oxidized samples by UV–visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).