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LC-MS/MS-based Monitoring of Protein Biotransformation


Wed, Jul 19, 2017 12:00 PM EDT


Nico van de Merbel

Prof.dr. Nico van de Merbel
Senior Director of Bioanalytical Science
PRA Health Sciences


Over the past decade, LC-MS/MS, the traditional workhorse for small-molecule bioanalysis, has been developed into a platform for protein quantification, as an alternative or addition to ligand-binding assays.

One of the interesting features of LC-MS/MS is its potential to give structural information and this enables the investigation of the in vivo biotransformation of biopharmaceuticals, a topic which so far has been relatively unexplored.

In this seminar, an LC-MS/MS approach is presented for the quantitative monitoring of the in vivo deamidation of the biopharmaceutical monoclonal antibody trastuzumab at a crucial position in its complementarity determining region (CDR). The multiplexed assay allows simultaneous quantification of five molecular species derived from trastuzumab after tryptic digestion: a stable peptide, a deamidation-sensitive peptide, two deamidated products and an intermediate.

Deamidation of trastuzumab at this position in the CDR was observed both in a 56-day in vitro forced degradation study and in plasma samples obtained from breast cancer patients after treatment with the drug for several months. Comparison with the results obtained by ELISA showed that deamidation of the drug gives rise to quantitative differences between the two techniques.


Key Learning Objectives:

  • How to apply LC-MS/MS for in vitro and in vivo biotransformation studies of biopharmaceuticals
  • How to optimize chromatography and detection for simultaneous quantification of multiple peptides
  • How to interpret the quantitative results obtained and how to compare them to ELISA data


Who Should Attend:

  • LC-MS and ELISA scientists working in the field of biopharmaceutical quantification in the pharmaceutical, contract-research and related industries
  • LC-MS scientists working in the field of small-molecule bioanalysis in the pharmaceutical, contract-research and related industries


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