ABSTRACT
Isomerization of aspartic acid residues is one of the major causes of chemical degradation during the shelf life of biological pharmaceuticals. Monoclonal antibody biopharmaceuticals are typically stored at mildly acidic pH conditions, which can lead to the isomerization reaction. The mechanism of this non-enzymatic chemical reaction has been studied in great detail. However, the identification and quantification of the isomerization sites in a given protein still remains a challenge. We developed an ion-pair reversed-phase HPLC method for the separation of an intact monoclonal antibody variant containing a single isoaspartic acid residue from its native counterpart. We identified and characterized the isomerization site using ion-pair reversed-phase HPLC mass spectrometry methods of the reduced and alkylated antibody and the enzymatically cleaved antibody. Lys-C followed by Asp-N digestion of the antibody was used for the identification of the isomerization site. Electron transfer dissociation (ETD) mass spectrometry was used to confirm the isomerization site at a DY motif at an aspartic acid residue in the CDR-H3 region of the antibody. Tyrosine at the C-terminus of an aspartic acid residue is typically not regarded as a hot spot for isomerization. Our findings suggest that it is not possible to predict isomerization sites in proteins with confidence and all aspartic acid residues located in the CDR regions of antibodies must be considered as potential isomerization site due to the solvent exposure or the flexibility of these regions of the molecule. Additionally, the effect of the pH on the isomerization rate was evaluated using the ion-pair reversed-phase HPLC method, showing that at a lower pH the isomerization rate is faster. Storage at 25°C for 6 months resulted in an increase of the amount of isoaspartic acid to 6.6% at pH 5.4, 6.0% at pH 5.8, and 5.6% at pH 6.2.
