Validation of Rapi-Fluor method for glycan profiling and application to commercial antibody drugs

Lim, Myung Sin; So, Min Kyung; Lim, Chung Su; Song, Du Hyun; Kim, Jong-Won; Woo, Jurang; Ko, Byoung Joon

doi:10.1016/j.talanta.2019.01.093

Cited by 23 publications

(26 citation statements)

References 27 publications

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“…The molecular weight of AG8 IgG was measured both with and without PNGase F treatment, and the accuracies were < 1 Da. The glycan profile of AG8 IgG was slightly different from that of the IgG standard, but the result was similar to those shown in other reports 27 , 28 . No analyzed physicochemical properties created an issue for subsequent in vitro and in vivo assays.…”

Section: Resultssupporting

confidence: 90%

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A human antibody against human endothelin receptor type A that exhibits antitumor potency

Ahn

Han

et al. 2021

Exp Mol Med

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Endothelin receptor A (ETA), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ETA nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ETA antibody (AG8) exhibiting high specificity for ETA in the β-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ETA using either a CHO-K1 cell line stably expressing human ETA or HT-29 colorectal cancer cells, in which AG8 exhibited IC50 values of 56 and 51 nM, respectively. In addition, AG8 treatment repressed the transcription of inhibin βA and reduced the ETA-induced phosphorylation of protein kinase B and extracellular regulated kinase. Furthermore, tumor growth was effectively inhibited by AG8 in a colorectal cancer mouse xenograft model. The human anti-ETA antibody isolated in this study could be used as a potential therapeutic for cancers, including colorectal cancer.

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Section: Resultssupporting

confidence: 90%

“…The injection concentration and volume were 0.1 mg/ml and 5 μl, respectively. Glycan profiling was performed with a Rapi-Fluor labeling kit (Waters, Milford, MA, USA), and all procedures were performed as described previously 27 , 28 .…”

Section: Methodsmentioning

confidence: 99%

A human antibody against human endothelin receptor type A that exhibits antitumor potency

Ahn

Han

et al. 2021

Exp Mol Med

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“…The isomers did separate in the LC with the F(6)A2G1 isomer eluting first as has been widely reported previously ( Figure S2). 33 The major species observed in the MS for each isomer was the [M+2H] 2+ species with an m/z 968. The CCS distributions (CCSD) of the [M+2H] 2+ species for each isomer was calculated and plotted to see if there were any noticeable differences ( Figure S3a & S3b).…”

Section: Resultsmentioning

confidence: 97%

“…We identified that the glycans from Immunoglobulin G (IgG) would be an ideal case study as it typically contains a mixture of two F(6)A2G1 isomers (Figure S1a & S1b). 33 These isomers differ by the position of a β1,4 galactose on either the α1,3 arm or the α1,6 arm of the glycan ( Figure S1). This mixture of isomers was ideal to assess whether glycan isomers could be identified in positive ion mode using IMS.…”

Section: Resultsmentioning

confidence: 99%

Ion-Mobility Mass Spectrometry of Glycans and Glycopeptides Using Both Intact and Glycan Sequencing Approaches In Positive Ion Mode Facilitates Glycan Isomer and Glycan Motif Identification

Pallister¹,

Choo²,

walsh³

et al. 2020

Preprint

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High resolution glycan analysis has become an important part of biopharmaceutical API production and quality control. Liquid chromatography (LC) is now a well-established technique in this field but the resolution of similar isomeric glycan structures is still a challenge. Here we show that the addition of ion mobility spectroscopy (IMS) in a hyphenated LC-IMS-MS setting allows for the high resolution of N-glycan isomers during positive ion analysis. We have identified unique features in the IM chromatograms to help differentiate a range of isomeric N-glycans for both RFMS labelled glycans and glycopeptides.<br>

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“…2-AA and 2-AB are both performing similarly well in positive-ion mode MS detection, with 2-AA likewise allowing sensitive negative-mode detection in both MALDI-and ESI-MS applications (Anumula & Dhume, 1998;Ruhaak et al, 2008;Reiding et al, 2017). An alternative group of tags that are specifically used for N-glycan analysis are the amine-reactive reagents which are targeting the glycosyl amine that is generated by PNGase F-mediated release of N-glycans (Lim et al, 2019). These tags (e.g., RFMS and procainamide; Figure 3) often combine fast, facile workflows with high sensitivity in fluorescence and MS detection (Keser et al, 2018).…”

Section: Hilic-and Rp-lc-ms Of Tagged Glycansmentioning

confidence: 99%

High sensitivity glycomics in biomedicine

Lageveen-Kammeijer

Küster

Reusch

et al. 2021

Mass Spectrometry Reviews

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Many analytical challenges in biomedicine arise from the generally high heterogeneity and complexity of glycan-and glycoconjugate-containing samples, which are often only available in minute amounts. Therefore, highly sensitive workflows and detection methods are required. In this review mass spectrometric workflows and detection methods are evaluated for glycans and glycoproteins. Furthermore, glycomic methodologies and innovations that are tailored for enzymatic treatments, chemical derivatization, purification, separation, and detection at high sensitivity are highlighted. The discussion is focused on the analysis of mammalian N-linked and GalNAc-type O-linked glycans.

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Validation of Rapi-Fluor method for glycan profiling and application to commercial antibody drugs

Cited by 23 publications

References 27 publications

A human antibody against human endothelin receptor type A that exhibits antitumor potency

A human antibody against human endothelin receptor type A that exhibits antitumor potency

Ion-Mobility Mass Spectrometry of Glycans and Glycopeptides Using Both Intact and Glycan Sequencing Approaches In Positive Ion Mode Facilitates Glycan Isomer and Glycan Motif Identification

High sensitivity glycomics in biomedicine

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