Reactivity-driven cleanup of 2-Aminobenzamide derivatized oligosaccharides

Chu, An-Hsiang Adam; Saati, Andrew; Scarcelli, John J.; Cornell, Richard J.; Porter, Thomas J.

doi:10.1016/j.ab.2018.01.012

Cited by 15 publications

(14 citation statements)

References 30 publications

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“…S2A). The liberated pentose was labeled with 2-aminobenzamide (2AB) 20 and excess free-2AB subsequently removed by treatment with octanal, aldehyde reagent 21 . This procedure facilitated the detection of small amounts of reaction products to clean up the remaining unreacted labeling reagents.…”

Section: Resultsmentioning

confidence: 99%

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Occurrence of a d-arabinose-containing complex-type free-N-glycan in the urine of cancer patients

Tanaka-Okamoto

Hanzawa

Murakami

et al. 2022

Sci Rep

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Urinary free-glycans are promising markers of disease. In this study, we attempted to identify novel tumor markers by focusing on neutral free-glycans in urine. Free-glycans extracted from the urine of normal subjects and cancer patients with gastric, colorectal, pancreatic and bile duct were fluorescently labeled with 2-aminopyridine. Profiles of these neutral free-glycans constructed using multidimensional high performance liquid chromatography separation were compared between normal controls and cancer patients. The analysis identified one glycan in the urine of cancer patients with a unique structure, which included a pentose residue. To reveal the glycan structure, the linkage fashion, monosaccharide species and enantiomer of the pentose were analyzed by high performance liquid chromatography and mass spectrometry combined with several chemical treatments. The backbone of the glycan was a monoantennary complex-type free-N-glycan containing β1,4-branch. The pentose residue was attached to the antennal GlcNAc and released by α1,3/4-l-fucosidase. Intriguingly, the pentose residue was consistent with d-arabinose. Collectively, this glycan structure was determined to be Galβ1-4(d-Araβ1-3)GlcNAcβ1-4Manα1-3Manβ1-4GlcNAc-PA. Elevation of d-arabinose-containing free-glycans in the urine of cancer patients was confirmed by selected reaction monitoring. This is the first study to unequivocally show the occurrence of a d-arabinose-containing oligosaccharide in human together with its detailed structure.

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Section: Resultsmentioning

confidence: 99%

“…The aqueous layer was separated by centrifugation, followed by the addition of chloroform and vigorous mixing. The aqueous layer was separated by centrifugation and filtered 21 . The 2AB labeled monosaccharides were separated using a Shim-pack Scepter C18-120 (2.1 × 150 mm, 3 μm; Shimadzu, Kyoto, Japan) at 55 °C.…”

Section: Methodsmentioning

confidence: 99%

Occurrence of a d-arabinose-containing complex-type free-N-glycan in the urine of cancer patients

Tanaka-Okamoto

Hanzawa

Murakami

et al. 2022

Sci Rep

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“…Advanced enrichment methods have also been explored to facilitate labeled glycan recovery. For example, Chu et al used reactivity driven dye clean-up protocols as an alternative for solid phase extraction (SPE) for glycan enrichment, where they specifically used octanal as liquid-liquid extraction/reaction solvent that showed minimal sample loss but allowed excess 2-AB removal 18 . Alternatively, Wu et al used bacterial cellulose as an efficient and low cost SPE enrichment matrix for rapid labeled glycan cleanup in under 10 minutes 19 .…”

Section: Introductionmentioning

confidence: 99%

An Integrated Process Analytical Platform for Automated Monitoring of Monoclonal Antibody N-linked Glycosylation

Gyorgypal

Chundawat

2021

Preprint

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The biopharmaceutical industry is transitioning towards adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Regulatory agencies such as the Food and Drug Administration (FDA) are further urging use of advanced PAT to analyze the design space to increase process knowledge and enable high quality biologics production. Post-translational modification of proteins, such as N-linked glycosylation are often critical quality attributes known to affect biologics safety and efficacy hence requiring close monitoring during manufacturing. Here, we developed an online sequential-injection based PAT system, called N-GLYcanyzer, that can rapidly monitor mAb glycosylation during upstream biomanufacturing. The key innovation includes design of an integrated mAb sampling and derivation system for antibody titer and glycoform analysis in under 2 hours. The N-GLYcanyzer process includes mAb capture, deglycosylation, fluorescent glycan labeling, and glycan enrichment for direct injection and analysis on an integrated high performance liquid chromatography (HPLC) system. Different fluorescent tags and reductants were tested to maximize glycan labeling efficiency under aqueous conditions, while porous graphitized carbon (PGC) was studied for optimum glycan recovery and enrichment. We find that 2-AB labeling of glycans with 2-picoline borane as a reducing agent, using the N-GLYcanyzer workflow, gives higher glycan labeling efficiency under aqueous conditions leading to upwards of a 5-fold increase in fluorescent products intensity. Finally, we showcase how the N-GLYcanyzer platform can be implemented at/on-line to an upstream bioreactor for automated and near real-time glycosylation monitoring of a Trastuzumab biosimilar produced by Chinese Hamster Ovary (CHO) cells.Graphical AbstractN-GLYcanyzer is an automated PAT toolkit for rapid sample processing for mAb N-linked glycans analysis to enable advanced biologics manufacturing

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“…Figure shows how modifications to the original octanal extraction procedure improve the removal of residual 2-AB. Figure A shows the residual fluorescence intensity that is obtained from unremoved 2-AB, following the original protocol in ref . In this case, the 2-AB labeling solution, containing no saccharide, was vortexed with 600 μL of octanal; octanal was discarded, and the fluorescence of the aqueous solution was measured.…”

Section: Resultsmentioning

confidence: 99%

“…To obtain a single peak corresponding to a particular glycan feature, saccharides are selectively cleaved with a glycosidase that releases the glycan of interest. Next, a standard fluorescent derivatization with 2-AB is used, and excess fluorophore is removed in a rapid, automatable fashion through the adaptation of a procedure first reported by Chu et al 15 Samples are transferred to a 384-well plate, and the fluorescence intensity is rapidly measured, an approach more conducive to high-throughput sample analysis than conventional glycan profiling. The fluorescent signal is specific to the glycan cleaved by the endoglycosidase or exoglycosidase used, allowing for the quantitation of specific features in a simple signal output.…”

Section: ■ Introductionmentioning

confidence: 99%

So You Discovered a Potential Glycan-Based Biomarker; Now What? We Developed a High-Throughput Method for Quantitative Clinical Glycan Biomarker Validation

2020

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Glycomic-based approaches to discover potential biomarkers have shown great promise in their ability to distinguish between healthy and diseased individuals; these methods can identify when aberrant glycosylation is significant, but they cannot practically be adapted into widely implemented diagnostic assays because they are too complex, expensive, and low-throughput. We have developed a new strategy that addresses challenges associated with sample preparation, sample throughput, instrumentation needs, and data analysis to transfer the valuable knowledge provided by protein glycosylation into a clinical environment. Notably, the detection limits of the assay are in the single-digit picomole range. Proof of principle is demonstrated by quantifying the changes in the sialic acid content in fetuin. As the sialic acid content in proteins varies in a number of disease states, this example demonstrates the utility of the method for biomarker analysis. Furthermore, the developed method can be adapted to other biologically important saccharides, affording a broad array of quantitative glycomic analyses that are accessible in a high-throughput, plate-reader format. These studies enable glycomic-based biomarker discovery efforts to transition through the difficult landscape of developing a potential biomarker into a clinical assay.

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Reactivity-driven cleanup of 2-Aminobenzamide derivatized oligosaccharides

Cited by 15 publications

References 30 publications

Occurrence of a d-arabinose-containing complex-type free-N-glycan in the urine of cancer patients

Occurrence of a d-arabinose-containing complex-type free-N-glycan in the urine of cancer patients

An Integrated Process Analytical Platform for Automated Monitoring of Monoclonal Antibody N-linked Glycosylation

So You Discovered a Potential Glycan-Based Biomarker; Now What? We Developed a High-Throughput Method for Quantitative Clinical Glycan Biomarker Validation

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