Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7μm sorbent

Ahn, Joomi; Bones, Jonathan; Yu, Ying; Rudd, Pauline M.; Gilár, Martin

doi:10.1016/j.jchromb.2009.12.013

Cited by 190 publications

(160 citation statements)

References 26 publications

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“…However, when site-specific information is essential, mass spectrometrybased methods might be more useful. 1,3,55,60,70 In the second part of our report, the mass spectrometry-based methods will be presented and an overall comparison between all methods will be given.…”

Section: Discussionmentioning

confidence: 99%

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

Reusch

Haberger

Maier

et al. 2015

mAbs

146

119

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(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.

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Section: Discussionmentioning

confidence: 99%

“…The supernatant was dried and reconstituted in an excess solution of 15 ml of acidic APTS (ProZyme) (5 mg in 0.5 ml of 15% v/v glacial acetic acid) and 5 ml of 1 M sodium cyanoborohydride in tetrahydrofurane (Aldrich). This solution was heated at 55 C for 2 h. The solution was diluted with water to a final volume of 250 ml.…”

Section: Ce-lif(apts-hr1)mentioning

confidence: 99%

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

Reusch

Haberger

Maier

et al. 2015

mAbs

146

119

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“…HILIC has already been applied in the past to the field of biopharmaceuticals for released glycan profiling and glycopeptide separations [8,9]. Wide-pore HILIC phases now offer new possibilities in glycan analysis at intact or middle-up levels of analysis [7,10].…”

Section: Introductionmentioning

confidence: 99%

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

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a b s t r a c tHydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

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“…The efficiency of silica particles improves with their decreasing size. Recently, fully porous sub-2 mm particles were introduced for UHPLC (ultra-high performance liquid chromatography) in the HILIC mode (Ahn et al, 2010;Lurie et al, 2011;. Better separation efficiency for polar and basic compounds on sub-2 mm coreeshell silica particles was reported, in comparison to fully porous particles of the same size, because of a reduced diffusion path inside the particles and consequently reduced sample transfer resistance (van Deemter C-coefficients) under HILIC conditions .…”

Section: Silica Gel and Hybrid Inorganic Sorbentsmentioning

confidence: 99%

Theory and Practice of UHPLC and UHPLC–MS

Guillarme

Veuthey

2017

Handbook of Advanced Chromatography /Mass Spectrometry Techniques

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Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7μm sorbent

Cited by 190 publications

References 26 publications

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

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

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Theory and Practice of UHPLC and UHPLC–MS

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