title: Isomerization of an aspartic acid residue in the complementarity-determining regions of a recombinant antibody to human IgE: identification and effect on binding affinity.
abstract: This report describes the effect on antigen binding of an isomerized aspartate residue located in the complementarity-determining regions (CDRs) of a recombinant monoclonal antibody. The antibody, which binds human IgE, contains two Asp-Gly sequences within its CDRs, but only one site was found to be labile to isomerization. Isolation and characterization of antibody fragments differing in the labile sequence were facilitated by using a technique involving hydrophobic interaction chromatography (HIC) that separates aspartyl, isoaspartyl, and cyclic imide variants to the residue located in CDR-L1. The variants were isolated for structural characterization and for determination of their relative antigen binding affinities. Mutants were constructed with altered residues to obviate the effects of isomerization and were evaluated for their ability to bind to IgE. Inspection of published crystal structures of CDRs of antibodies indicated that hydrogen binding of the Asp side chain of the unreactive residue may be the constraint that prevents isomerization. The strategy outlined here may prove to be of general utility in the biochemical and immunochemical characterization of recombinant antibodies.
authors.affiliation: Department of Manufacturing Sciences, Genentech Inc., South San Francisco, California 94080, USA.
authors.forename: J
authors.initials: J
authors.lastname: Cacia
authors.forename: R
authors.initials: R
authors.lastname: Keck
authors.forename: L G
authors.initials: LG
authors.lastname: Presta
authors.forename: J
authors.initials: J
authors.lastname: Frenz
mesh_terms: D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000915:Antibody Affinity; D001224:Aspartic Acid / Q000737:chemistry; D014644:Genetic Variation; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D001224:Aspartic Acid; D007073:Immunoglobulin E