Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

摘要: Although LC−MS methods are increasingly used for the absolute quantification of proteins, lack appropriate internal standard (IS) hinders development rapid and standardized analytical both in vitro vivo studies. Here, we have developed a novel method therapeutic protein, which is monoclonal antibody (mAb). The combines liquid chromatography tandem mass spectrometry (LC−MS/MS) protein cleavage isotope dilution with isotope-labeled mAb as IS. latter was identical to analyzed exception that each threonine contains four 13C atoms one 15N atom. Serum samples were spiked IS prior overnight trypsin digestion subsequent sample cleanup. Sample extracts on C18 ACE column (150 mm × 4.6 mm) using an LC gradient time 11 min. Endogenous concentrations determined by calculating peak height ratio its signature peptide corresponding isotop...