High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

Reusch, Dietmar; Haberger, Markus; Kailich, Tobias; Heidenreich, Anna-Katharina; Kampe, Michael; Bulau, Patrick; Wuhrer, Manfred

doi:10.4161/mabs.26712

Cited by 57 publications

(46 citation statements)

References 50 publications

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“…28,[39][40][41][42] All other methods applied fluorescent labeling. Four methods used electrophoretic separation, which included conventional high-resolution capillary gel electrophoresis with laser-induced fluorescence [CE-LIF(APTS-HR1)], [43][44][45][46][47][48] DNA-sequencer-aided fluorophore-assisted carbohydrate electrophoresis after 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling for high-throughput screening [DSA-FACE(APTS)], [49][50][51][52][53][54] high-resolution capillary gel electrophoresis with rapid labeling with APTS via reductive amination [CE-LIF (APTS-HR2)], and cartridge-based capillary gel electrophoresis with rapid 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) labeling, in development specifically for screening [CCGE(ANTS)]. The CE methods differ with regard to the labeling method: APTS labeling for the "normal" method requires 4 to 24 h for labeling, whereas rapid reductive amination has been optimized Three laboratories were involved in performing the experiments, an analytical laboratory in a development department, a quality control laboratory and a laboratory of a vendor of glycoanalytical tools.…”

Section: Resultsmentioning

confidence: 99%

“…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. In addition, online ESI-MS(/MS) coupling of the HILIC-UHPLC separation of the APTS-labeled mAb1 N-glycans was employed for further structural elucidation.…”

Section: Peak Assignmentmentioning

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

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146

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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: Resultsmentioning

confidence: 99%

Section: Peak Assignmentmentioning

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

Self Cite

146

119

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show abstract

“…Separation is then commonly achieved by hydrophilic interaction liquid chromatography (HILIC)-HPLC or UPLC [16,17], or by C(G)E with fluorescence detection [18,19]. As presented in a multilaboratory study, fluorescent labeling is the critical step in profiling due to possible incomplete derivatization, resulting in greater laboratory variability than MS-based Nglycomic methods [20].…”

Section: Current Methods In Glycosylation Analysis Of Biopharmaceuticalsmentioning

confidence: 99%

Mass spectrometry for glycosylation analysis of biopharmaceuticals

Dotz

Haselberg

Shubhakar

et al. 2015

TrAC Trends in Analytical Chemistry

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“…This approach allowed for routine analysis of well-characterized glycans with the more in-depth characterization of minor glycans utilizing MS analysis. Reusch et al [100] also took a combined method approach but introduced a DNA analyzer for fluorescence detection. APTS-labeled glycans were initially identified with hydrophilic interaction liquid chromatography-ultra performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS) analysis and subsequent capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) with a DNA analyzer.…”

Section: Combined Analytical Techniques To Attain Comprehensive Charamentioning

confidence: 99%

Characterization of Pharmaceutical IgG and Biosimilars Using Miniaturized Platforms and LC-MS/MS

Wooding¹,

Peng²,

Mechref

2016

CPB

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Therapeutic monoclonal antibodies (mAbs) have made a tremendous impact in treating patients with various diseases. MAbs are designed to target specifically a cell and illicit a response from the immune system to destroy the cell. As originator mAb drug patents are coming to an end, generic pharmaceutical companies are poised to replicate and produce so-called biosimilar drugs. MAbs are significantly more complicated than small drugs to analyze and produce. The mAb proteoform and glycoform must be as similar to the original drug as possible to be a viable replacement. The mAb proteoform is well characterized but can be altered through various undesirable reactions such as deamidation. The mAb glycoform is harder to replicate as the glycan formation is a complicated template-less one; it is proving difficult for the originator companies to produce a homogenous population of mAbs from batch to batch. Severe side-effects have occurred in patients taking mAbs with immunogenic glycans, highlighting the importance of quality control mechanisms. The complex nature of mAbs requires sensitive and robust tools amenable to the high-throughput analysis required by a manufacturing setting. Miniaturized analytical platforms for complex biosimilar analysis are still in their infancy but have shown great promise for sample preparation. Capillary electrophoresis-laser induced fluorescence remains a powerful and fast technique for routine glycan analysis. Mass spectrometry is the method of choice for the analysis of mAb proteoforms and is emerging as a powerful tool for glycoform analysis.

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High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

Cited by 57 publications

References 50 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

Mass spectrometry for glycosylation analysis of biopharmaceuticals

Characterization of Pharmaceutical IgG and Biosimilars Using Miniaturized Platforms and LC-MS/MS

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