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

Bults, P., Bischoff, R., Bakker, H., Gietema, J. A. & van de Merbel, N. C., 11-Feb-2016, In : Analytical Chemistry. 88, 3, p. 1871-1877 7 p.

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  • LC-MS-MS-based monitoring of in vivo protein biotransformation

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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 three hours 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 international bioanalytical guidelines over the clinically relevant range of 0.5 to 500 µg/mL with bias and CV values well below 15%. Deamidation of trastuzumab was observed in plasma both in a 56-day in vitro forced degradation study (up to 37% of the total drug concentration) and in samples obtained from breast cancer patients after treatment with the drug for several months (up to 25%). Comparison with a validated ELISA method for trastuzumab showed that deamidation of the drug at the CDR leads to a loss of recognition by the antibodies used in the ELISA assay.

Original languageEnglish
Pages (from-to)1871-1877
Number of pages7
JournalAnalytical Chemistry
Issue number3
Publication statusPublished - 11-Feb-2016



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