Separation methods hyphenated to mass spectrometry for the characterization of the protein glycosylation at the intact level

Abstract

Glycosylation is one of the most common post-translational modifications of proteins that affects their biological activity, solubility, and half-life. Therefore, its characterization is of great interest in proteomic, particularly from a diagnostic and therapeutic point of view. However, the number and type of glycosylation sites, the degree of site occupancy and the different possible structures of glycans can lead to a very large number of isoforms for a given protein, called glycoforms. The identification of these glycoforms constitutes an important analytical challenge. Indeed, to attempt to characterize all of them, it is necessary to develop the development and optimization of the separation step to achieve high resolution between isoforms, the recent ones are much more application-oriented, such as clinical diagnosis, quality control, and glycoprotein monitoring in formulations or biological samples.

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

“…For example, the quantification of low abundant PTMs such as glycated and oxidized amino acids at the protein intact level might not be confirmed at the peptide level due to the lower sensitivity of the MAM approach (84). In addition, the analysis at the peptide level usually requires off-line and timeconsuming sample preparation procedures, which may generate artifacts such as oxidation, isomerization, and deamidation (85)(86)(87). To limit possible artificial degradation, Wang et al…”

“…D'Atri et al described a middle-up approach to compare the glycosylation patterns of originators and biosimilars mAbs by using HILIC coupled to HRMS (108). The HILIC mode was the most suitable for the elucidation of glycosylation patterns, due to the high resolving power and compatibility with MS (87,120,121). Similar strategies were successfully applied for the characterization of an ADC(122), Brentuximab Vedotin, and a recombinant Fc fusion protein, Etanercept (107).…”

Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches

“…10 Direct infusion electrospray ionization (ESI) MS can be used for intact glycoform analysis of systems with relatively low levels of glycosylation (such as monoclonal antibodies built on IgG templates), 10 although it can certainly benefit from incorporating an online separation step prior to MS analysis. 11 Hydrophilic interaction chromatography (HILIC) remains the most popular option for the glycoform separation prior to the MS step; 12,13 however, it requires the use of mobile phases with a high organic solvent content, inevitably leading to the glycoprotein denaturation. Apart from being problematic for proteins having low tolerance to organic co-solvents, this also creates challenges for the analysis of large oligomeric proteins in which variation of the number of polypeptide subunits within the protein molecule is another source of intrinsic heterogeneity (in addition to glycosylation).…”

Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS

“…With 8 potential glycosylation sites, hCG is a highly heterogeneous protein and the characterization of all its glycoforms constitutes an important analytical challenge. The analysis of the protein at the intact level by liquid chromatography (LC) or capillary electrophoresis (CE) hyphenated with mass spectrometry (MS) is a fast and easy approach that allows the determination of the glycosylation profile [6]. Recently, we optimized for the first time a CE method hyphenated with MS for the separation of hCG glycoforms [7].…”