title: Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain.
abstract: We investigated the formation and stability of succinimide, an intermediate of deamidation events, in recombinant monoclonal antibodies (mAbs). During the course of an analytical development study of an IgG1 mAbs, we observed that a specific antibody population could be separated from the main product by cation-exchange (CEX) chromatography. The cell-based bioassay measured a approximately 70% drop in potency for this fraction. Liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) and tandem mass spectrometry (LC-MS/MS) analyses showed that the modified CEX fraction resulted from the formation of a succinimide intermediate at Asn 55 in the complementarity determining region (CDR) of the heavy chain. Biacore assay revealed a approximately 50% decrease in ligand binding activity for the succinimide-containing Fab with respect to the native Fab. It was found that the succinimide form existed as a stable intermediate with a half-life of approximately 3 h at 37 degrees C and pH 7.6. Stress studies indicated that mildly acidic pH conditions (pH 5) favored succinimide accumulation, causing a gradual loss in potency. Hydrolysis of the succinimide resulted in a further drop in potency. The implications of the succinimide formation at Asn 55, a highly conserved residue among IgG1 (mAbs), are discussed.
authors.affiliation: Department of Analytical and Formulation Science, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119, USA. byan@amgen.com
authors.forename: Boxu
authors.initials: B
authors.lastname: Yan
authors.forename: Sean
authors.initials: S
authors.lastname: Steen
authors.forename: David
authors.initials: D
authors.lastname: Hambly
authors.forename: John
authors.initials: J
authors.lastname: Valliere-Douglass
authors.forename: Tim
authors.initials: T
authors.lastname: Vanden Bos
authors.forename: Scott
authors.initials: S
authors.lastname: Smallwood
authors.forename: Zac
authors.initials: Z
authors.lastname: Yates
authors.forename: Thomas
authors.initials: T
authors.lastname: Arroll
authors.forename: Yihong
authors.initials: Y
authors.lastname: Han
authors.forename: Himanshu
authors.initials: H
authors.lastname: Gadgil
authors.forename: Ramil F
authors.initials: RF
authors.lastname: Latypov
authors.forename: Alison
authors.initials: A
authors.lastname: Wallace
authors.forename: Aiching
authors.initials: A
authors.lastname: Lim
authors.forename: Gerd R
authors.initials: GR
authors.lastname: Kleemann
authors.forename: Weichun
authors.initials: W
authors.lastname: Wang
authors.forename: Alain
authors.initials: A
authors.lastname: Balland
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D022801:Complementarity Determining Regions / Q000737:chemistry*; D006207:Half-Life; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D008024:Ligands; D013058:Mass Spectrometry; D010206:Papain / Q000737:chemistry; D010449:Peptide Mapping; D013388:Succinimides / Q000138:chemical synthesis*; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D008024:Ligands; D013388:Succinimides; D001216:Asparagine; D014357:Trypsin; D010206:Papain