Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry

A technique for rapid characterization of variable regions of monoclonal antibodies (mAb) is described. Several intact mAbs were analyzed on a Thermo-Fisher LTQ-Orbitrap high-resolution mass spectrometer (MS) by in-source fragmentation. In-source fragmentation has the unique advantage of fragmenting all charge states of a protein at the same time and, thus, greatly improves the sensitivity of the fragment ions over a true MS/MS experiment, where a single charge state is isolated and fragmented. In addition, immediate fragmentation of the protein before tertiary structure formation may also facilitate protein fragmentation. This technique has been proved very useful for top-down analysis of large proteins. In-source fragmentation of mAbs generated a series of fragment ions. In addition to some small b and y ions from the light chain and heavy chain in the low m/z region, a series of b ions corresponding to N-terminal 106-120 residues of both heavy chain and light chain were observed. The cleavage sites for these b ions happen to be near the linker regions between the variable domains and the constant domains of these antibodies. These b ions, therefore, correspond to the entire variable region of each chain. Similar results were obtained for all mAbs analyzed, including both immunoglobulin G1 and G2 molecules. To further characterize the variable regions, these b ions were isolated and fragmented by collision-induced dissociation in the linear trap, followed by mass analysis in the orbitrap. Large number of product ions was observed from these b ions. Many of these product ions are internal fragments between the two disulfide-linked cysteine residues. To demonstrate the capability of the technique, several mAbs were force-oxidized by treating with tert-butyl hydroperoxide, followed by mass spectrometric analysis. In-source fragmentation and MS/MS of the variable region b ions clearly identified the locations of the oxidized methionine.