title: Identification and quantification of degradations in the Asp-Asp motifs of a recombinant monoclonal antibody.
abstract: Two degradations of aspartate residues located in Asp-Asp motifs in the CDR3 region of a recombinant monoclonal antibody were identified and quantified after the antibody was aged in a mildly acidic buffer at elevated temperatures. The degraded sample aged at 25 degrees C for 1 month generated 1.8% antibody molecules that had isomerization in the aspartate residues, while the degraded sample after aging at 45 degrees C for 1 month contained 7% isomerization. Peptide bond cleavages at the aspartate residues were also detected and characterized. The percentage of clipped antibody molecules after 1 month of storage was 1% at 25 degrees C and 4.4% at 45 degrees C. The generated cleaved polypeptides were noncovalently attached to the intact antibody molecule and were not involved in the aggregation formation. They were not detected by native size-exclusion chromatography because of their strong non-covalent association to the rest of the antibody molecules. On the other hand, the cleaved polypeptides were dissociated and detected as fragments under denaturing conditions of reversed-phase HPLC, denaturing size-exclusion chromatography and MALDI-TOF mass spectrometry. It was demonstrated that the cleavages at the above aspartate residue sites occurred due to the aging of the sample at elevated temperatures and were not method-induced by the reversed-phase HPLC and other methods used in this study.
authors.affiliation: Amgen, Department of Pharmaceutics, One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Xiao
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Bondarenko
mesh_terms: D020816:Amino Acid Motifs; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D022801:Complementarity Determining Regions; D019281:Dimerization; D004355:Drug Stability; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D011994:Recombinant Proteins