Monitoring papain digestion of a monoclonal antibody by electrospray ionization mass spectrometry.
Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the Fab, F(ab')2 and deglycosylated Fc fragments obtained from the murine IgG1 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 Fab and F(ab')2 subunits indicated that the predominant site of papain cleavage occurred at C221 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 C221 from the C-terminal end of the Fd subunit. ESI analysis of the cleaved Fab 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 Fd 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 (M81 and M136). Removal of the carbohydrate portion from the Fc fragment by N-glycosidase F indicated that papain cleavage had occurred at C223 of the B72.3 MAb heavy chain. In addition, it was observed that the C-terminal lysine residue (K438) was absent from the deglycosylated Fc 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 well as providing a rapid and sensitive technique for assessing MAb fragments prior to use in radioimaging or radiotherapy.
Bennett
K L
KL
Department of Chemistry, University of Wollongong, NSW, Australia.
Smith
S V
SV
Truscott
R J
RJ
Sheil
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D006031:Glycosylation; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism*; D010449:Peptide Mapping; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D010206:Papain