Monitoring papain digestion of a monoclonal antibody by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the F-ab, F(ab')(2) and deglycosylated F-c fragments obtained from the murine IgG(1) B72.3 monoclonal antibody (MAb) by digestion with the sulfhydryl protease papain, in an attempt to determine the sites of cleavage and thus to clarify the mode of action of this enzyme on MAbs. ESI analysis of the F-ab and F(ab')(2) subunits indicated that the predominant site of papain cleavage occurred at C-221 of the B72.3 MAb heavy chain. Reduction of the intra- and interchain disulfide bridges of these fragments by 1,4-dithiothreitol and subsequent electrospray analysis showed a loss of C-221 from the C-terminal end of the Fd subunit. ESI analysis of the cleaved F-ab fragment indicated that there was an apparent loss of amino acid residues from this fragment. Edman sequencing of the cleaved subunit revealed an intact light chain and the loss of QVQ from the N-terminal of the F-d subunit. Reduction of this subunit gave a Fd fragment approximately 32 Da greater than the predicted mass, which we have attributed to oxidation of the heavy chain methionine residues (M(81) and M(136)). Removal of the carbohydrate portion from the F-c fragment by N-glycosidase F indicated that papain cleavage had occurred at C-223 of the B72.3 MAb heavy chain. In addition, it was observed that the C-terminal lysine residue (K-438) was absent from the deglycosylated F-c fragment, presumably due to carboxypeptidase B activity that occurs during the in vivo production of the B72.3 MAb in murine hosts. These data clearly illustrate the power of ESI-MS for determining small changes in mass on large proteins as web as providing a rapid and sensitive technique for assessing MAb fragments prior to use in radioimaging or radiotherapy. (C) 1997 Academic Press.