title: Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule.
abstract: Deamidation of asparagine residues, a post-translational modification observed in proteins, is a common degradation pathway in monoclonal antibodies (mAbs). The kinetics of deamidation is influenced by primary sequence as well as secondary and tertiary folding. Analytical hydrophobic interaction chromatography (HIC) is used to evaluate hydrophobicity of candidate mAbs and uncover post-translational modifications. Using HIC, we discovered atypical heterogeneity in a highly hydrophobic molecule (mAb-1). Characterization of the different HIC fractions using LC/MS/MS revealed a stable succinimide intermediate species localized to an asparagine-glycine motif in the heavy chain binding region. The succinimide intermediate was stable in vitro at pH 7 and below and increased on storage at 25°C and 40°C. Biacore evaluation showed a decrease in binding affinity of the succinimide intermediate compared with the native asparagine molecule. In vivo studies of mAb-1 recovered from a pharmacokinetic study in cynomolgus monkeys revealed an unstable succinimide species and rapid conversion to aspartic/iso-aspartic acid. Mutation from asparagine to aspartic acid led to little loss in affinity. This study illustrates the importance of evaluating modifications of therapeutic mAbs both in vitro and in serum, the intended environment of the molecule. Potential mechanisms that stabilize the succinimide intermediate in vitro are discussed.
authors.affiliation: AbbVie Bioresearch Center; Worcester, MA USA.
authors.forename: David
authors.initials: D
authors.lastname: Ouellette
authors.affiliation: AbbVie Bioresearch Center; Worcester, MA USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Chumsae
authors.affiliation: AbbVie Bioresearch Center; Worcester, MA USA.
authors.forename: Anca
authors.initials: A
authors.lastname: Clabbers
authors.affiliation: AbbVie Bioresearch Center; Worcester, MA USA.
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Radziejewski
authors.affiliation: AbbVie Bioresearch Center; Worcester, MA USA.
authors.forename: Ivan
authors.initials: I
authors.lastname: Correia
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000915:Antibody Affinity; D001216:Asparagine / Q000097:blood / Q000737:chemistry; D001666:Binding Sites, Antibody; D002845:Chromatography; D018604:Epitope Mapping; D005998:Glycine / Q000097:blood / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000097:blood / Q000737:chemistry*; D007167:Immunotherapy; D066298:In Vitro Techniques; D008252:Macaca fascicularis; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D013388:Succinimides / Q000097:blood / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D013388:Succinimides; C032620:succinimide; D001216:Asparagine; D005998:Glycine
keywords: IgG1; aspartate; deamidation; iso-aspartate; monoclonal antibody; succinimide