Determination of Antibody Glycosylation by Mass Spectrometry

Abstract

Immunoglobulin (Ig) G is formed by two antigen-binding moieties termed Fabs and a conserved Fc -portion, which interacts with components of the immune systems. Within the Fc, N-linked carbohydrates are attached to each conserved asparagine residue at position 297 within the CH2 domain. These oligosaccharide moieties introduce a higher degree of heterogeneity within the molecule, by influencing stability of the antibody and its mediated effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). The carbohydrate moieties can vary strongly depending on the production host and can be manipulated by different fermentation conditions, thereby influencing the function of the antibody. Therefore it is necessary to carefully monitor changes in the carbohydrate composition during cell line development and production processes. This chapter describes two different mass spectrometry based methods used for analyses of the carbohydrate moieties attached to the Fc-part of human IgG1. In the first approach, the glycans are released from the antibody by endoglycosidase (Peptide N Glycosidase F) digestion and monitored by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS), whereas in the second method the carbohydrate structures, still attached to an enzymatically produced Fc-fragment, are analyzed by electrospray ionization mass spectrometry.

A new tool for monoclonal antibody analysis

“…Compared with the aforementioned proteases, IdeS provides a few advantages, including high site specificity, simple and robust digestion procedure, and high yield. [17][18][19] Application of IdeS has been demonstrated in many literature reports on IgG1 and IgG2 molecules, [20][21][22][23][24][25][26][27][28] as well as Fc fusion proteins. 29,30 Almost all the reports to date used primarily LC-MS techniques in subsequent analysis of the resulting antibody fragments.…”

“…[20][21][22][23][24][25]30,33,34,63 In most reported studies, antibody fragments were analyzed by LC-MS techniques directly to determine glycoform distributions. Whereas MS-based methods are superior in speed and sample consumption, the complexity in operating MS instruments and in data analysis often prevent them from being used in routine quality tests.…”

3.5 Manipulation of Cell Growth, Metabolism and Product Quality Attributes

“…While the level of N-and O-glycan galactosylation has not yet been conclusively linked to major effects on pharmaceutical efficacy of glycoprotein drug substance, it still has become a quality attribute of therapeutic glycoproteins that is closely monitored during bioprocessing [151][152][153][154]. The future will tell whether major deviations from the galactosylation quality target product profile will be tolerated for lot release and which galactosylation profile of production lots will be considered to be out of compliance with approved process specifications.…”

Glycosylation analysis of Chinese hamster ovary produced glycoproteins

“…Common derivatization procedures are similar to those used for HPLC or HILIC as listed above, in addition to permethylation [111] and labeling based on hydrazone linkages [112]. Instruments implemented with MS/MS can provide information for fine structural assignment of oligosaccharides through characteristic fragmenta tion patterns [113,114]. There are several software pack ages that perform automated interpretation of MS/MS spectra of glycans; however, it is advised to complement the analysis by rigorous visual inspection of the data, as subtleties may have been overlooked by algorithms.…”