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(A) Bar chart summarises the relative abundance (in percentage) of glycans of EEQY N STYR and EEQY N STYR (only those with relative abundance higher than 0.5% are plotted). Relative abundance of (B) fucosylation, (C) sialylation, (D) galactosylation, and (E) bisecting structure of serum IgG. (A), Two-way ANOVA followed by Bonferroni’s post hoc test, multiple comparisons were performed between EEQY N STYR and EEQY N STYR, n = 4; (B–E), Student’s t-test; n = 4, *p < 0.05, **p < 0.01; ****p < 0.0001).
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The glycosylation of antibody-based proteins is vital in translating the right therapeutic outcomes of the patient. Despite this, significant infrastructure is required to analyse biologic glycosylation in various unit operations from biologic development, process development to QA/QC in bio-manufacturing. Simplified mass spectrometers offer ease o...
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... Thus, many glycoinformatic softwares were developed to accelerate glycan and glycopeptide analysis [111]. For example, coupling GlycopeptideGraphMS, a software which uses graph theory to cluster glycan LC-MS data into grouped networks, combined with a compact LC-MS system enabled semi-automatic, rapid, and accurate analysis of human serum IgG and mAbs glycopeptides [112]. The performance of this workflow is comparable to those performed with the traditional glycan analytical workflow (i.e. ...
The circulatory half-life of recombinant therapeutic proteins is an important pharmacokinetic attribute because it determines the dosing frequency of these drugs, translating directly to treatment cost. Thus, recombinant therapeutic glycoproteins such as monoclonal antibodies have been chemically modified by various means to enhance their circulatory half-life. One approach is to manipulate the N-glycan composition of these agents. Among the many glycan constituents, sialic acid (specifically, N-acetylneuraminic acid) plays a critical role in extending circulatory half-life by masking the terminal galactose that would otherwise be recognised by the hepatic asialoglycoprotein receptor (ASGPR), resulting in clearance of the biotherapeutic from the circulation. This review aims to provide an illustrative overview of various strategies to enhance the pharmacokinetic/pharmacodynamic properties of recombinant therapeutic proteins through manipulation of their sialic acid content.
... Released N-glycans were analysed as previously described [22]. First, 10 µL of reconstituted released N-glycans were injected into an ACQUITY H-Class UPLC (Waters Corporation, Milford, MA, USA) coupled to a SYNAPT XS mass spectrometer (Waters Corporation, Milford, MA, USA). ...
It is estimated that food fraud, where meat from different species is deceitfully labelled or contaminated, has cost the global food industry around USD 6.2 to USD 40 billion annually. To overcome this problem, novel and robust quantitative methods are needed to accurately characterise and profile meat samples. In this study, we use a glycomic approach for the profiling of meat from different species. This involves an O-glycan analysis using LC-MS qTOF, and an N-glycan analysis using a high-resolution non-targeted ultra-performance liquid chromatography-fluorescence-mass spectrometry (UPLC-FLR-MS) on chicken, pork, and beef meat samples. Our integrated glycomic approach reveals the distinct glycan profile of chicken, pork, and beef samples; glycosylation attributes such as fucosylation, sialylation, galactosylation, high mannose, α-galactose, Neu5Gc, and Neu5Ac are significantly different between meat from different species. The multi-attribute data consisting of the abundance of each O-glycan and N-glycan structure allows a clear separation between meat from different species through principal component analysis. Altogether, we have successfully demonstrated the use of a glycomics-based workflow to extract multi-attribute data from O-glycan and N-glycan analysis for meat profiling. This established glycoanalytical methodology could be extended to other high-value biotechnology industries for product authentication.
The therapeutic and immunological properties of biopharmaceuticals are governed by the glycoforms contained in them. Thus, bioinformatics tools capable of performing comprehensive characterization of glycans are significantly important to the biopharma industry. The primary structural elucidation of glycans using MS is tricky and tedious in terms of spectral interpretation. In this study, the biosimilars of a therapeutic monoclonal antibody and an Fc‐fusion protein with moderate and heavy glycosylation, respectively, were employed as representative biopharmaceuticals for released glycan analysis using LC‐MS/MS instead of conventional MS‐based analysis. SimGlycan® ‐ a software with proven ability to process tandem MS data for released glycans could identify eight additional glycoforms in Fc‐fusion protein biosimilar, which were not detected during MS analysis of released glycans or glyco‐peptide mapping of the same molecule. Thus, LC‐MS/MS analysis of released glycans not only complements conventional LC‐MS‐based glycan profiling but can also identify additional glycan structures that may otherwise be omitted during conventional LC‐MS based analysis of mAbs. The MS data processing tools, such as PMI Byos™ and SimGlycan® etc, can display pivotal analytical capabilities in automated LC‐MS and LC‐MS/MS‐based glycan analysis workflows, especially for high‐throughput structural characterization of glycoforms in biopharmaceuticals. This article is protected by copyright. All rights reserved
