An Integrated Strategy for Structural Characterization of the Protein and Carbohydrate Components of Monoclonal Antibodies: Application to Anti-Respiratory Syncytial Virus MAb

Abstract

The relatively rapid and extensive characterization of the amino acid sequence and site-specific carbohydrate structures of a recombinant, reshaped human monoclonal antibody directed against respiratory syncytial virus (RSHZ19) is presented. The integrated strategy used a combination of mass spectrometric and conventional methodologies. Liquid chromatography/electrospray mass spectrometry was used for peptide mapping and selective identification of glycopeptides, and Edman degradation and tandem mass spectrometry were used to define the sequences of selected peptides. Matrix-assisted laser desorption/ionization mass spectrometry provided the M(r) of the intact protein and was used to characterize endo- and exoglycosidase digests of isolated glycopeptides to identify the glycosylation-site peptide and define the structures of the 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 heterogeneity at the N and C termini of the heavy chain were determined. Oxidation of a specific methionine residue to the sulfoxide was demonstrated by sequencing the N-terminally blocked peptide by tandem MS. Carbohydrate was found exclusively at Asn296 of the heavy chain. There was no evidence for a nonglycosylated form of the molecule or for the presence of O-linked carbohydrate. The qualitative distribution of glycoforms at this site was determined by MS of the isolated, tryptic glycopeptide and compared with results obtained by high-performance anion exchange chromatography and high-resolution gel permeation chromatography of oligosaccharides released by hydrazinolysis. The sequence and linkage of individual glycan species were determined using matrix-assisted laser desorption/ionization MS to monitor the results of a series of controlled digestions with specific exoglycosidases. The set of glycoforms consists predominantly of biantennary, core fucosylated carbohydrates lacking sialic acid. The present study is one of the first to directly evaluate the quantitative as well as qualitative consistency of the MS methods with conventional methods for carbohydrate analysis.

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

“…However, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) [7,8], ESI orthogonal-TOF [9,10] and ESI quadrupole [2,[11][12][13] mass spectrometers have been the instruments of choice for MAbs and macromolecules, largely due to the large m/z range of the TOF and high ion transmission efficiency of the quadrupole mass analyzers. Unfortunately, on-line RP-HPLC has not accompanied these previous studies of intact monoclonal antibodies.…”

Identification and characterization of glycosylation sites in human serum clusterin

“…The relative amounts of glycoforms listed in Table 5 reflect the approximate attachment site-specific distribution of oligosaccharides among those detected without adjustment for partial occupancy. It is important to emphasize that MS can only be used to provide an estimate of the relative amounts of glycoforms, and considerable caution must be exercised in deriving and using these values (see Roberts et al, 1995). Because serum clusterin represents the reservoir of clusterin synthesized in a number of tissues (including but not limited to liver, kidney, lung, and heart), it is possible that specific oligosaccharide structures may be found associated with clusterin depending upon tissue source, physiological status, disease state, and species.…”

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

“…Alternatively, protein molecular mass analysis is relatively fast, does not require lengthy sample preparation, and induces fewer, if any, modifications [8] compared with the peptide mapping [5,7]. Analysis of intact monoclonal IgG antibodies and their large domains has been reported for matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) sources and almost all mass analyzers including MALDI-time of flight (TOF) [9 -11], ESI quadrupole (Q) [4,[12][13][14], ion trap [15], orthogonal TOF [8,11,16,17], and the LTQ-Orbitrap during direct infusion [18]. Although a mass change in a population of mAbs can be used to monitor post-translational modifications [8], it cannot reveal the site of modification.…”