HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals
Abstract
In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Chromatographic techniques such as HILIC and capillary electrophoresis have been used together with fluorescence detection of glycans to obtain quantitative glycomic profiles.
“Chromatographic techniques such as hydrophilic interaction chromatography (HILIC) and capillary electrophoresis continue to improve and have also been used together with fluorescence detection of glycans to obtain quantitative glycomic profiles, however these methods are hindered in their ability to assign glycan structures to specific signals [143,144].”
From Glycoengineering Chinese hamster ovary cells: a short history (2021) · cites this paper as [143]
HILIC and capillary electrophoresis with fluorescence detection are hindered in their ability to assign glycan structures to specific signals.
“Chromatographic techniques such as hydrophilic interaction chromatography (HILIC) and capillary electrophoresis continue to improve and have also been used together with fluorescence detection of glycans to obtain quantitative glycomic profiles, however these methods are hindered in their ability to assign glycan structures to specific signals [143,144].”
From Glycoengineering Chinese hamster ovary cells: a short history (2021) · cites this paper as [143]
No citation lists for this paper
No citation lists for this paper yet — checked again daily.
References 23 · Cited by 104
- Authors
- Melmer, M., Stangler, T., Schiefermeier, M., Brunner, W., Toll, H., Rupprechter, A., Lindner, W., & Premstaller, A.
- Affiliations
- University of Vienna (AT)
- Corresponding author
- Premstaller, Andreas
- Venue
- Analytical and Bioanalytical Chemistry
- Published
- 2010-07-17
- Volume
- 398
- Issue
- 2
- Pages
- 905-914
- ISSN
- 1618-2642 (ISSN-L)
- 1618-2650
- OpenAlex
- W1998653677
- Cited by
- 104
- References
- 23
- Citations by year
- 2026: 5 · 2025: 4 · 2024: 4 · 2023: 4 · 2022: 7 · 2021: 12 · 2020: 8 · 2019: 5 · 2018: 9 · 2017: 3 · 2016: 4 · 2015: 10 · 2014: 10 · 2013: 7 · 2012: 3
- FWCI
- 2.34
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Analytical and Bioanalytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Life Sciences › Immunology and Microbiology › Immunology › Biosimilars and Bioanalytical Methods
- Keywords
- N-glycans, protein glycosylation, biopharmaceuticals, hydrophilic interaction liquid chromatography, glycan heterogeneity, glycan characterization, quality control, glycopeptides
- MeSH terms
- Carbohydrate Sequence, Chromatography, High Pressure Liquid, Chromatography, Liquid, Fluorescent Dyes, Glycoproteins, Molecular Sequence Data, Polysaccharides, Sensitivity and Specificity, Mass Spectrometry, Reproducibility of Results, Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase, ortho-Aminobenzoates
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
