Sequential digestions enable identification and quantification of rapid aspartic acid isomerization in the CDR of a...

Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) within the complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs) can lead to conformational changes that decrease antigen-binding affinity. Although isomerization can significantly alter the chromatographic and electrophoretic profiles, precise localization of this modification requires a mass spectrometry-based approach, such as peptide mapping. In this work, we present a case study that investigates various analytical strategies to identify the root cause of significant changes observed in the chromatographic and electrophoretic profiles of an mAb during formulation development. LC-MS analysis of reduced mAb using high-resolution mass spectrometry, peptide mapping using trypsin digestion, and fraction collection of the newly identified peak followed by trypsin digestion suggested that isomerization occurs within the CDR of the mAb. However, due to the presence of three Asp residues within a single tryptic peptide, this modification could not be precisely localized. To overcome this limitation, we developed a sequential enzymatic digestion strategy, utilizing trypsin followed by Asp-N digestion, which enabled accurate localization and quantification of the isomerization site. The resulting data indicated that the main isoAsp signal originated from isomerization at the DS motif that increased substantially over time in the liquid formulation, while no significant change was observed in the lyophilized formulation. The level of isomerization determined through this sequential digestion method correlated well with the LC-UV quantitation data of the reduced mAb.