Identification of deamidation and isomerization sites on pharmaceutical recombinant antibody using H(2)(18)O

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 H(2)(18)O 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 (18)O is incorporated into the protein molecule. The mAb was lyophilized and reconstituted in H(2)O or H(2)(18)O, followed by incubation at 50 degrees 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 ( approximately 3Da) at Asn was observed in the newly produced peptide when the mAb was incubated in H(2)(18)O, whereas it was barely feasible to identify this mass shift in H(2)O. In the other peptide fragment, isomerization of Asp was identified after incubation in H(2)(18)O, although it was impossible to distinguish when using H(2)O. 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.