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

Abstract

(2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 1: Separation-based methods, mAbs, 7:1, 167-179, DOI: 10.4161/19420862.2014.986000 To link to this article: https://doi.org/10. 4161/19420862.2014 Abbreviations: mAb, monoclonal antibody; Fc, fragment crystallizable; IgG, immunoglobulin G; HILIC-UPLC, hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography; 2-AB, 2-aminobenzamide; Fab, fragment, antigen-binding; CE-LIF, capillary electrophoresis-laser induced fluorescence; HPLC, high performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption/ionization-mass spectrometry; ESI-MS, electrospray ionization-mass spectrometry; HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection; APTS, 8-aminopyrene-1, 3, 6-trisulfonic acid; DSA-FACE, DNA-sequencer-aided fluorophore-assisted carbohydrate electrophoresis; ANTS, 8-aminonaphthalene-1, 3, 6-trisulfonate; CCGE, cartridge-based capillary gel electrophoresis; HR, high resolution; IAB, InstantAB labeling; CHO, Chinese hamster ovaryImmunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fcglycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.

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

“…Table 1 compiles the results of CE-ESI-MS and HILIC-FD (2-AB) relative abundance values obtained for each mAb. As described in the literature [20], HILIC-FD shows excellent precision with low standard deviations (with the exception of Infliximab-Remsima® analysis). The suggested CE-ESI-MS method also presents low absolute variation with values below 4% for the different glycan structures.…”

“…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.…”

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

“…22 Derivatization may be used to neutralize and stabilize sialic acids and enable sialic acid analysis by MS. 23 We recently published a comparison of non-MS, separation-based methods for glycan analysis of a mAb by enzymatic glycan release followed by HPLC or capillary electrophoresis (CE)-separation after fluorescent labeling, or by HPAEC-PAD. 24 Here, we describe a thorough comparison of MSbased methods for glycan analysis using the same mAb sample. The study involved 2 laboratories, a biopharmaceutical company (Roche Diagnostics GmbH), and an academic research laboratory (Leiden University Medical Center).…”

Ultrafast and high-throughput N-glycan analysis for monoclonal antibodies

“…The 2-AB HILIC-HPLC method is the mainstay for glycan analysis for recombinant antibodies in the biopharmaceutical industry. However, the method is time consuming (requiring several hours with recently improved labeling chemistry or up to three days using older methods 35 ) and labor intensive, and becomes a bottleneck in the analysis of large numbers of samples.…”