ENZYMES IMMOBILIZATION ON MONOLITHIC DISKS: AN INNOVATIVE STRATEGY FOR PROTEIN DIGESTION AND DRUG METABOLISM.

Trypsin is the most used proteolytic enzyme for protein digestion and Peptide Mass Fingerprinting (PMF). Digestions can be performed in solution with classical incubations protocols of 4-24 h. However, in the search for faster and automated methodologies, an interesting alternative is the use of solid phase digestion achieved by immobilizing trypsin on methacrylate-based monolithic CIM (Convective Interaction Media®) disks. Rapid on-line digestion (5 min) of numerous proteins was achieved by inserting the trypsin-based IMER (Immobilized Enzyme Reactor) in a liquid chromatography system hyphenated to (or with?) tandem mass spectrometry (IMER-LC-MS/MS). On-line results were compared in terms of sequence coverage with classical in-solution digestions and commercially available Poroszyme® cartridge. Presenting high performances in terms of digestion time, long term stability and costs, the monolithic IMER demonstrated to be an attractive tool to perfor rapid and automated protein digestions. One of the main reasons for new chemical entities failure in drug discovery could be attributed to inadequate pharmacokinetic properties. Therefore, the determination of major metabolic pathways and the study of drug-drug interactions are mandatory for optimizing suitable drug candidates. In this context, ethylenediamine (EDA) CIM® minidisks were selected for non-covalent immobilization of recombinant-expressed CYP2D6 and CYP3A4 liposomes (RECO System®). Resulting CYP450-based IMERs were integrated in a fully automated IMER-LC-MS/MS for on-line phase I drug metabolism studies with probe substrates and a known competitive inhibitor. Results showed that the optimized immobilization procedure was suitable to maintain the enzyme activity and that the inclusion of CYPs in the IMER significantly