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Rapid N -glycan release from glycoproteins using immobilized PNGase F microcolumns
- Source :
- Journal of Chromatography B. 1032:139-143
- Publication Year :
- 2016
- Publisher :
- Elsevier BV, 2016.
-
Abstract
- N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.
- Subjects :
- 0301 basic medicine
PNGase F
Glycan
Glycosylation
Immobilized enzyme
Recombinant Fusion Proteins
Clinical Biochemistry
01 natural sciences
Biochemistry
Analytical Chemistry
law.invention
03 medical and health sciences
chemistry.chemical_compound
Ribonucleases
N-linked glycosylation
Polysaccharides
law
Escherichia coli
Animals
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Fetuins
Glycoproteins
chemistry.chemical_classification
Chromatography
biology
010401 analytical chemistry
Electrophoresis, Capillary
Cell Biology
General Medicine
Enzymes, Immobilized
Fetuin
0104 chemical sciences
030104 developmental biology
chemistry
Immunoglobulin G
biology.protein
Recombinant DNA
Glycoprotein
Subjects
Details
- ISSN :
- 15700232
- Volume :
- 1032
- Database :
- OpenAIRE
- Journal :
- Journal of Chromatography B
- Accession number :
- edsair.doi.dedup.....d3cc3f12d03758aa577cb4a0ead5d23e