Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

Du, Yi; May, Kimberly; Xu, Wei; Liu, Hongcheng

doi:10.1007/s13361-012-0397-8

Cited by 10 publications

(7 citation statements)

References 39 publications

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“…The method used to measure fucosylation of antibodies is similar to that in a previously described report (53). The mAb was reduced (45°C, 30 min, 4.5 M Gn-HCl, 5 mM DTT).…”

Section: Methodsmentioning

confidence: 99%

Development of orally active inhibitors of protein and cellular fucosylation

Okeley

Alley

Anderson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

158

188

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The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDPfucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.

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“…The method used to measure fucosylation of antibodies is similar to that in a previously described report (53). The mAb was reduced (45°C, 30 min, 4.5 M Gn-HCl, 5 mM DTT).…”

Section: Methodsmentioning

confidence: 99%

Development of orally active inhibitors of protein and cellular fucosylation

Okeley

Alley

Anderson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

158

188

View full text Add to dashboard Cite

show abstract

“…Therefore many combinations of these classes of glycoform can lead to the same overall afucosylation content. Further complicated arise from the fact that some afucosylated Fc glycans are undetectable by certain analytical methods so that method dependent considerations can be important [5]. It is common to match afucosylation content by simply comparing the relative amounts of the major classes of glycoform along with the overall afucosylation content.…”

Section: Discussionmentioning

confidence: 99%

Afucosylated Biosimilars need Afucosylation Specific Assays

Chung¹,

Zhan²

2017

MOJCSR

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“…Specifically in glycoproteomics, LC is advantageous as it can be coupled to ESI for the separation of complex samples prior to online MS analysis. This approach is extensively used to map glycoprotein biomarkers 105,106,107 , as well as to analyze glycans on recombinant monoclonal antibodies 108,109 .…”

Section: Separation and Derivatization Techniques 141 Chromatographymentioning

confidence: 99%

Qualitative and Quantitative Assessment on the Use of Magnetic Nanoparticles for Glycopeptide Enrichment

Bodnar

Perreault

2013

Anal. Chem.

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Glycoproteomics represent the field of study of the dynamic changes occurring among glycoconjugates within the cellular compartments. Changes in glycosylation have been linked to various diseases, including metastatic carcinomas in which the 9 carbon sialic acid moiety has been shown to play a prominent role. The common method used to study these aberrant changes most often includes a mass spectrometer at some stage in the workflow. However, serum samples contain many proteins which inhibit the analysis of these glycosylation changes, and ergo, enrichment steps are employed as a measure to help alleviate this ailment. Routinely, this is accomplished using lectins, either alone or in combination, to retrieve proteins with specific sugar linkages within the serum sample. This methodology, although known to be very specific, requires many washing steps, making it a cumbersome addition to a high throughput workflow. Presented here is an alternative protocol using custom-made amine functionalized magnetic nanoparticles (MNP) which are nearly 4× smaller than those used before for similar purposes. The developed protocol is based on both hydrophilic interaction and weak anion exchange principles, allowing it to target glycopeptides but, more specifically, those which contain sialylation. For quantification purposes, tandem mass tags from Thermo Scientific were utilized to compare the enrichment efficiencies between the magnetic nanoparticle method and a commercially available glycopeptide enrichment kit offered through EMD Millipore. The MNP method is fast (~10 min) and simple and can quantitatively and qualitatively enrich sialylated glycopeptides more than the commercially available kit.

show abstract

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

Cited by 10 publications

References 39 publications

Development of orally active inhibitors of protein and cellular fucosylation

Development of orally active inhibitors of protein and cellular fucosylation

Afucosylated Biosimilars need Afucosylation Specific Assays

Qualitative and Quantitative Assessment on the Use of Magnetic Nanoparticles for Glycopeptide Enrichment

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