title: Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies.
abstract: The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.
authors.affiliation: Late Stage Pharmaceutical and Device Development Group, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA. wakankar.aditya@gene.com
authors.forename: Aditya A
authors.initials: AA
authors.lastname: Wakankar
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Borchardt
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.forename: Steven
authors.initials: S
authors.lastname: Shia
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Shire
authors.forename: Jun L
authors.initials: JL
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D022801:Complementarity Determining Regions / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D010446:Peptide Fragments / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D010446:Peptide Fragments; D001224:Aspartic Acid