AN INTEGRATED STRATEGY FOR STRUCTURAL CHARACTERIZATION OF THE PROTEINAND CARBOHYDRATE COMPONENTS OF MONOCLONAL-ANTIBODIES - APPLICATION TOANTI-RESPIRATORY SYNCYTIAL VIRUS MAB

The relatively rapid and extensive characterization off the amino acid sequence and site-specific carbohydrate structures of a recombinant, reshaped human monoclonal antibody directed against respiratory syncyt ial virus (RSHZ19) is presented. The integrated strategy used a combin ation of mass spectrometric and conventional methodologies, liquid chr omatography/electrospray mass spectrometry was used for peptide mappin g and selective identification of glycopeptides, and Edman degradation and tandem mass spectrometry were used to define the sequences of sel ected peptides. Matrix-assisted laser desorption/ionization mass spect rometry provided the M(r) of the intact protein and was used to charac terize endo- and exoglycosidase digests of isolated glycopeptides to i dentify the glycosylation-site peptide and define the structures of th e carbohydrates at that site. These experiments verified 99.1% of the light- and 99.3% of the heavy-chain amino acid sequences. The N and C termini of both chains were confirmed, and the nature and extent of he terogeneity at the N and C termini of the heavy chain were determined. Oxidation of a specific methionine residue to the sulfoxide was demon strated by sequencing the N-terminally blocked peptide by tandem MS. C arbohydrate was found exclusively at Asn(296) of the heavy chain, Ther e was no evidence for a nonglycosylated form of the molecule or for th e presence of O-linked carbohydrate. The qualitative distribution of g lycoforms at this site was determined by MS of the isolated, tryptic g lycopeptide and compared with results obtained by high-performance ani on exchange chromatography and high-resolution gel permeation chromato graphy of oligosaccharides released by hydrazinolysis. The sequence an d linkage of individual glycan species were determined using matrix-as sisted laser desorption/ionization MS to monitor the results of a seri es of controlled digestions with specific exoglycosidases. The set of glycoforms consists predominantly of biantennary, core fucosylated car bohydrates lacking sialic acid. The present study is one of the first to directly evaluate the quantitative as well as qualitative consisten cy of the MS methods with conventional methods for carbohydrate analys is.