Pre-clinical estimation of cetuximab using nano-surface and molecular orientation limited (nSMOL) proteolysis and LC-MS/MS

nSMOL works on selective proteolysis of Fab region by making use of difference in size of the protease nanoparticle diameter (200 nm) and the antibody resin pore size (100 nm). To achieve limited proteolysis of the antibody Fab region, the antibody is immobilized in such a way that only Fab region of antibody is spatially available for selective cleavage with protease (trypsin) immobilized on beads. Moreover, effective Fab proteolysis is possible under non-denaturing physiological condition. Thus, there is a considerable reduction of the digested peptides that are formed which enhances selectivity and drastically reduces time required for method setup and optimization. In other words, using nSMOL, one can maintain the specificity of the antibody sequences while minimizing the sample complexity as well as the elimination of extra protease.