Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

Abstract

(2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: Mass spectrometric methods, mAbs, 7:4, 732-742, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

“…The suggested CE-ESI-MS method also presents low absolute variation with values below 4% for the different glycan structures. These values are comparable to those determined for other MS-based methods, such as NanoLC-ESI-MS described elsewhere [21]. It is worth noticing that for each mAb, the deviations were obtained based on the combination of digestions and injections performed in triplicates by different experimenters over an extended period of several weeks, thus the results strongly support the performance of the method in terms of robustness and reproducibility.…”

“…This behaviours mean that the detected sum of mono-antennary structures were not over-estimated. Moreover, this result confirmed that during ESI-MS analysis of glycopeptides, in source decay can efficiently be avoided through the proper choice of the MS conditions and voltages, even for CE-ESI-MS method [21]. The sum of afucosylated species (G0, G1, G2) is a relevant parameter for antibody effector function.…”

“…4.65 (<0.1) fragmentation of bi-antennary structures resulting in the loss of one antenna can generate elevated mono-antennary structures levels, along with a charge reduction that is commonly observed in MS spectra and that is a consequence of a loss of the N-acetylglucosamine [21]. While only low or similar levels of mono-antennary structures were detected in CE-ESI-MS for eight mAbs, higher levels of these glycovariants were detected for natalizumab and nivolumab, as compared to HILIC-FD (Table S2).…”

“…The complexity and heterogeneity of the glycosylation pattern is mainly due to mAbs production in living expression systems [14][15][16] and requires a number of orthogonal analytical techniques to be fully characterized. Several analytical methods have been described for the glyco-variants characterization at different levels (from released glycans to intact protein level) including separative techniques (liquid chromatography (LC), capillary electrophoresis (CE)) often coupled to spectrometric, amperometric and mass spectrometric detection [17][18][19][20][21]. Recently, Reusch's group published two major articles dealing with the analysis of Fc-glycosylation profiles, and comparing several separation methods hyphenated or not with mass spectrometry (MS) detection [20,21].…”

“…Several analytical methods have been described for the glyco-variants characterization at different levels (from released glycans to intact protein level) including separative techniques (liquid chromatography (LC), capillary electrophoresis (CE)) often coupled to spectrometric, amperometric and mass spectrometric detection [17][18][19][20][21]. Recently, Reusch's group published two major articles dealing with the analysis of Fc-glycosylation profiles, and comparing several separation methods hyphenated or not with mass spectrometry (MS) detection [20,21]. This comprehensive comparison showed an excellent precision and accuracy for all the methods.…”