Mass spectrometry of the humanized monoclonal antibody CAMPATH 1H.
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.
Ashton
D S
DS
Wellcome Research Laboratories, Beckenham, Kent, U.K.
Beddell
C R
CR
Cooper
D J
DJ
Craig
S J
SJ
Lines
A C
AC
Oliver
R W
RW
Smith
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D003412:Cricetulus; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D007141:Immunoglobulin Fc Fragments / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011134:Polysaccharides / Q000032:analysis
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D011134:Polysaccharides