title: Unveiling a glycation hot spot in a recombinant humanized monoclonal antibody.
abstract: Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.
authors.affiliation: Protein Analytical Chemistry, Cell Culture Development, Purification Development, Protein Chemistry, and Antibody Engineering, Genentech, Inc., South San Francisco, California 94080, USA. bzhang@gene.com
authors.forename: Boyan
authors.initials: B
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Inn
authors.initials: I
authors.lastname: Yuk
authors.forename: Roger
authors.initials: R
authors.lastname: Pai
authors.forename: Patrick
authors.initials: P
authors.lastname: McKay
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.forename: Mark
authors.initials: M
authors.lastname: Dennis
authors.forename: Viswanatham
authors.initials: V
authors.lastname: Katta
authors.forename: Kathleen Champion
authors.initials: KC
authors.lastname: Francissen
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins