title: Cysteinylation of a monoclonal antibody leads to its inactivation.
abstract: Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism.
authors.affiliation: a Abbott Diagnostic R & D, Abbott Diagnostics Division, Abbott Laboratories , Abbott Park , IL , USA.
authors.forename: Troy
authors.initials: T
authors.lastname: McSherry
authors.affiliation: a Abbott Diagnostic R & D, Abbott Diagnostics Division, Abbott Laboratories , Abbott Park , IL , USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: McSherry
authors.affiliation: a Abbott Diagnostic R & D, Abbott Diagnostics Division, Abbott Laboratories , Abbott Park , IL , USA.
authors.forename: Panfilo
authors.initials: P
authors.lastname: Ozaeta
authors.affiliation: b AbbVie Inc. , North Chicago , IL , USA.
authors.forename: Kenton
authors.initials: K
authors.lastname: Longenecker
authors.affiliation: a Abbott Diagnostic R & D, Abbott Diagnostics Division, Abbott Laboratories , Abbott Park , IL , USA.
authors.forename: Carol
authors.initials: C
authors.lastname: Ramsay
authors.affiliation: a Abbott Diagnostic R & D, Abbott Diagnostics Division, Abbott Laboratories , Abbott Park , IL , USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Fishpaugh
authors.affiliation: a Abbott Diagnostic R & D, Abbott Diagnostics Division, Abbott Laboratories , Abbott Park , IL , USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Allen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000276:immunology; D003545:Cysteine / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D011499:Protein Processing, Post-Translational / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D003545:Cysteine
keywords: Antibody; biacore; cysteinylation; electrospray ionization mass spectrometry; hydrophobic interaction chromatography; molecular modeling