MASS-SPECTROMETRY OF THE HUMANIZED MONOCLONAL-ANTIBODY CAMPATH 1H

Two mass spectrometric techniques, electrospray ionization (ESI) and m atrix-assisted laser desorption ionization (MALDI) have been used to s tudy the intact humanized monoclonal antibody CAMPATH 1H, its fully an d partially deglycosylated species, and 13 fragments prepared from it. The transformed ESI mass spectra of the glycosylated species gave com plex patterns of molecular masses (M(r)'s). These have been substantia lly assigned to the presence of a mixture of glycoforms, each resultin g from the combination of a single protein species with specific glyca ns of four distinct masses. The MALDI mass spectra of the glycosylated species, with the exception of that of the smallest fragment Fc/2, wh ich indicated the presence of three of the glycans, gave single M(r) v alues comparable to the mean M(r) calculated from the ESI results. The M(r) values for the 10 prepared nonglycosylated species sup port the validity of the published amino acid sequence for the antibody and def ine the cleavage sites for the enzymic fragmentations. It is concluded that mass measurement of the Fc/2 fragment using ESI techniques provi des a convenient means of preliminary assessment of the major glycosyl ated entities.