Mass Spectrometry of the Humanized Monoclonal Antibody Campath 1H

Abstract

Two mass spectrometric techniques, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) have been used to study the intact humanized monoclonal antibody CAMPATH 1H, its fully and partially deglycosylated species, and 13 fragments prepared from it. The transformed ESI mass spectra of the glycosylated species gave complex patterns of molecular masses (M(r's). These have been substantially assigned to the presence of a mixture of glycoforms, each resulting from the combination of a single protein species with specific glycans of four distinct masses. The MALDI mass spectra of the glycosylated species, with the exception of that of the smallest fragment Fc/2, which indicated the presence of three of the glycans, gave single M(r) values comparable to the mean M(r) calculated from the ESI results. The M(r) values for the 10 prepared nonglycosylated species support the validity of the published amino acid sequence for the antibody and define the cleavage sites for the enzymic fragmentations. It is concluded that mass measurement of the Fc/2 fragment using ESI techniques provides a convenient means of preliminary assessment of the major glycosylated entities.

Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry

“…The peak identities were therefore determined based on the molecular weights. Peak A contained antibody with two glycosylated heavy chains with typical oligosaccharides observed for recombinant monoclonal antibodies expressed in mammalian cell cultures [16][17][18][19]. Peak B contained antibodies with either, zero, one or two non-glycosylated heavy chains.…”

Heterogeneity of Monoclonal Antibodies

“…1), and various truncated structures with the loss of the core fucose, GlcNAc, or both. 48,63,[113][114][115][116][117][118][119][120][121][122][123][124][125][126][127] Oligosaccharides of high mannose have also been reported for several antibodies. 33,62,97,103,111,[128][129][130][131] Sialic acid is normally present at a very low level for Fc oligosaccharides.…”

Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells

“…Nterminal blocked heavy chain by a pyroglutamic acid (PyroGlu) resulting from the N-terminal glutamine cyclization and loss of ammonia (−17 Da) have been described for other IgG, [31][32][33][34][35] as well as for peptides [36,37].…”