LC-MS/MS-Based Monitoring of in vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and its Deamidation Products in Human Plasma

Bults et al., Anal Chem. (2016) - PMID: 26713683

Product(s) used in this publication:  Absolutely Quantified Peptides SpikeTides™ TQL


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 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.

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