Identification of deamidation and isomerization sites on pharmaceutical recombinant antibody using H218O

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are spontaneous and common alterations occurring in pharmaceutical protein drugs in solution. Because those reactions may cause functional changes, it is important to identify the product-related substances, especially when biopharmaceuticals are under development. In this study, we used H218O to identify Asn deamidation and Asp isomerization sites on a recombinant humanized monoclonal antibody (mAb) by using high-performance liquid chromatography–mass spectrometry (HPLC–MS). This strategy takes advantage of reactions whereby 18O is incorporated into the protein molecule. The mAb was lyophilized and reconstituted in H2O or H218O, followed by incubation at 50 °C for 1 month. Samples were reduced/carboxymethylated and digested by trypsin and then subjected to HPLC–MS and HPLC–tandem mass spectrometry (MS/MS) analysis. Among all of the peptide fragments analyzed, there were two in which deamidation and/or isomerization was observed. In one peptide fragment, an obvious mass shift (∼3 Da) at Asn was observed in the newly produced peptide when the mAb was incubated in H218O, whereas it was barely feasible to identify this mass shift in H2O. In the other peptide fragment, isomerization of Asp was identified after incubation in H218O, although it was impossible to distinguish when using H2O. By means of this procedure, identification of deamidation and isomerization sites can be accomplished easily even when they are difficult or impossible to detect by the usual peptide mapping.