title: LC-MS/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma.
abstract: 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.
authors.affiliation: Bioanalytical Laboratory, PRA Health Sciences, Early Development Services, Amerikaweg 18, 9407 TK Assen, The Netherlands.|Analytical Biochemistry, Department of Pharmacy, University of Groningen , A. Deusinglaan 1, 9700 AV Groningen, The Netherlands.
authors.forename: Peter
authors.initials: P
authors.lastname: Bults
authors.affiliation: Analytical Biochemistry, Department of Pharmacy, University of Groningen , A. Deusinglaan 1, 9700 AV Groningen, The Netherlands.
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
authors.affiliation: Bioanalytical Laboratory, PRA Health Sciences, Early Development Services, Amerikaweg 18, 9407 TK Assen, The Netherlands.
authors.forename: Hilde
authors.initials: H
authors.lastname: Bakker
authors.affiliation: Faculty of Medical Sciences, Department of Medical Oncology, University Medical Centre Groningen , Hanzeplein 1, 9713 GZ Groningen, The Netherlands.
authors.forename: Jourik A
authors.initials: JA
authors.lastname: Gietema
authors.affiliation: Bioanalytical Laboratory, PRA Health Sciences, Early Development Services, Amerikaweg 18, 9407 TK Assen, The Netherlands.|Analytical Biochemistry, Department of Pharmacy, University of Groningen , A. Deusinglaan 1, 9700 AV Groningen, The Netherlands.
authors.forename: Nico C
authors.initials: NC
authors.lastname: van de Merbel
mesh_terms: D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D015394:Molecular Structure; D013388:Succinimides / Q000097:blood* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D000068878:Trastuzumab / Q000097:blood* / Q000378:metabolism
chemical_list: D013388:Succinimides; C032620:succinimide; D000068878:Trastuzumab