Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

Abstract

a b s t r a c tThe IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)-UPLC, reversed phase (RP)-UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC-UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

“…33 For HPAEC-PAD, peaks were identified by spiking commercially available glycan standards and by employing exoglycosidase digests. Additionally the peaks were confirmed by online desalting and coupling to ESI-MS. 60 For CE-LIF(APTS-HR1), peaks were assigned by online coupling to ESI-MS as described by Gennaro et al 43 For DSA-FACE(APTS), assignment of peaks is described in Reusch et al 53 Briefly, glycan identification relied on the use of commercially available glycan standards (after APTS labeling) used to spike the DSA-FACE(APTS) analysis of APTS-labeled mAb1 glycans after HILIC-UHPLC fractionation.…”

“…33 Detected glycosylation features All the methods facilitated separation of the main Fc N-glycan species that are typically found on therapeutic IgG mAbs produced in Chinese hamster ovary (CHO) cells (G0F, G1F, G2F, G0, G1 and M5; see Table 2 for key). The Reference Method allowed the resolution and quantitation of 15 glycan species (Fig.…”

“…These amounts were not optimized for the lowest amounts of mAb1. Adamczyk et al 33 estimated the detection limit of APTS-labeled glycans by CE-LIF to be 0.4 nM. No special special skills for the analyst are needed; Investment: a HPLC system that is suited for the high-pH buffers and equipped with a pulsed amperometric detector…”

“…In principle the methods can be sub-divided into 3 categories: 3,[21][22][23] (1) Analysis of the IgG molecule with electrospray ionization mass spectrometry (ESI-MS)-either on the intact molecule after reduction of disulphide bonds, or after a limited digestion with a proteolytic enzyme and deduction of the overall glycan composition; [24][25][26] (2) Enzymatic release of the Fc glycans and measurement with mass spectrometric methods, by HPLC with pulsed amperometric detection or by capillary electrophoresis (CE)/ HPLC-based methods after fluorescent labeling; 27,28 and (3) Proteolytic cleavage of the IgG molecule and analysis of the glycopeptides with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization-mass spectrometry ESI-MS. [29][30][31][32] Comparisons of different methods for analysis of IgG Fc-glycosylation have been reported, but these studies included a limited number of methods or compared mainly mass spectrometry-based methods. [33][34][35][36][37][38] Thus, a thorough comparison of different methods for glycoanalysis is still lacking.…”

EndoSd: An IgG Glycan Hydrolyzing Enzyme in Streptococcus Dysgalactiae Subspecies Dysgalactiae

“…Fluorescence detection was achieved using excitation and emission wavelengths of 330 and 420 nm, respectively. The obtained chromatograms were compared with previously published and assigned IgG glycoforms [35,36]. The sequence, composition and linkage specificities of the glycans were analyzed by exoglycosidase digestion arrays (Prozyme, CA, USA).…”