Identification and measurement of isoaspartic acid formation in the complementarity determining region of a fully human monoclonal antibody

Isomerization plays a key role in protein degradation. This isomerization is often difficult to detect by many protein characterization methods such as SDS-PAGE, SEC, and IEF. This work shows the identification of an isomerized aspartic acid residue in the CDR2 of the heavy chain of a fully human monoclonal antibody. This isoaspartic acid increases significantly with storage at 2–8 °C. Hydrophobic interaction chromatography was utilized to separate the isoaspartic variant in the intact state. Mass spectrometry including peptide mapping was employed to identify and confirm the exact location of the modification. Since this modification occurs in the complementarity determining region (CDR) it was found that binding is reduced. Therefore, three different analytical methods for regular analysis of this isomerization are evaluated. These methods include peptide mapping by LC–MS, HIC, and a protein isoaspartate methyltransferase assay. It was determined that HIC is the best method to regularly assay the level of isomerization in this monoclonal antibody.