title: Cysteine racemization on IgG heavy and light chains.
abstract: Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of D-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible β-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide.
authors.affiliation: From the Department of Process and Product Development, Amgen, Inc., Thousand Oaks, California 91320.
authors.forename: Qingchun
authors.initials: Q
authors.lastname: Zhang
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Flynn
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry* / Q000276:immunology; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011499:Protein Processing, Post-Translational; D013440:Sulfides / Q000097:blood / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D013440:Sulfides; C029702:prolactin-binding protein; D003545:Cysteine
keywords: Antibodies; Chirality; Disulfide; Light Chain; Mass Spectrometry (MS); Post Translational Modification; Protein Chemical Modification; Thioether