Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping.
N-lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout i...
| Publicado en: | BioMed Research International Vol. 2018; pp. 1 - 11 |
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| Autores principales: | , , |
| Formato: | research tables/charts Journal Article |
| Publicado: |
Wiley-Blackwell
3/29/2018
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=128752458&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 128752458 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 23146133 FT2T jtl: BioMed Research International issn: 23146133 maglogo: N pubinfo: dt: 3/29/2018 vid: 2018 pid: 480 pub: Wiley-Blackwell place: Malden, Massachusetts artinfo: ui: 128752458 128752458 128752458 10.1155/2018/3909674 128752458 ppf: 1 ppct: 10 formats: fmt: – @attributes: type: T – @attributes: type: P tig: atl: Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping. aug: au: Eckard, Anahita D. Dupont, David R. Young, Johnie K. affil: Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA 94080, USA sug: subj: Glycosylation Polysaccharides Chromatography Methods Chemistry, Analytical Human Electrophoresis Peptide Mapping ab: N-lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N-glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography–mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for<italic> N</italic>-glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit,<italic> N</italic>-glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area. pubtype: Academic Journal doctype: research tables/charts Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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