Identification and quantification of degradations in the Asp-Asp motifs of a recombinant monoclonal antibody.

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.