LC-MS/MS-based monitoring of in vivo protein biotransformation: quantitative determination of trastuzumab and its deamidation products in human plasma

An LC-MS/MS-based method is described for quantitatively monitoring the in vivo deamidation of the biopharmaceutical monoclonal antibody trastuzumab at a crucial position in its complementarity determining region (CDR). The multiplexed LC-MS/MS assay using selected reaction monitoring (SRM) allows simultaneous quantitation of five molecular species derived from trastuzumab after tryptic digestion: a stable signature peptide (FTISADTSK), a deamidation-sensitive signature peptide (IYPTNGYTR), its deamidated products (IYPTDGYTR and IYPTisoDGYTR), and a succinimide intermediate (IYPTsuccGYTR). Digestion of a 50 μL plasma sample is performed at pH 7 for 3 h at 37 °C, which combines a reasonable (>80%) digestion efficiency with a minimal (<1%) formation of deamidation products during digestion. Rapid in vitro deamidation was observed at higher pH, leading to a (large) overestimation of the concentrations of deamidation products in the original plasma sample. The LC-MS/MS method was validated in accordance with ...