Bioanalysis Zone

LC-MS/MS-based quantitative monitoring of protein biotransformation





The multiplexed LC-MS/MS assay 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-uL 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. 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.

What will you learn?

  • How to use LC-MS/MS to simultaneously quantitate five molecular species of a biotherapeutic mAb
  • How to quantitatively monitor in vivo deamidation of the mAb
  • How the LC-MS/MS method compares to ELISA

Who may this interest?

  • CROs, pharmaceutical and biotechnology companies focused on LC-MS/MS quantitative bioanalysis of large molecules
  • Mass spectrometrists and bioanalytical chemists
  • Academicians and students interested in LC-MS/MS bioanalysis


Nico-BZsiteNico van de Merbel
Director of Bioanalytical Science
PRA Health Sciences

Nico van de Merbel is Director of Bioanalytical Science at the bioanalytical laboratories of PRA Health Sciences in Assen, The Netherlands, and Lenexa (KS, USA). His field of expertise is the bioanalytical application of chromatographic methods for small and large molecules and he is responsible for research, method development and the regulatory aspects of bioanalysis. Nico obtained his MSc in toxicology and his PhD in analytical chemistry at the Free University in Amsterdam, The Netherlands. He holds an honorary professorship in Industrial Bioanalysis at the University of Groningen, The Netherlands. He has over 25 years of experience in quantitative bioanalysis in academia and industry and has been employed with Pharma Bio-Research/PRA Health Sciences since 1995.

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