Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns

Marton Szigeti, Judit Bondar, Douglas Gjerde, Zsolt Keresztessy, Akos Szekrenyes, A. Guttman

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 10 min 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.

Original languageEnglish
Pages (from-to)139-143
Number of pages5
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1032
DOIs
Publication statusPublished - Oct 1 2016

Fingerprint

Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Glycosylation
Polysaccharides
Glycoproteins
Clone Cells
Carbohydrates
Fetuins
Mannose
Glutathione Transferase
Product development
Digestion
Industry
Immunoglobulin G
Robots
Liquids
Enzymes

Keywords

  • Capillary electrophoresis
  • Enzyme immobilization
  • N-glycosylation
  • PNGase F

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

Cite this

Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns. / Szigeti, Marton; Bondar, Judit; Gjerde, Douglas; Keresztessy, Zsolt; Szekrenyes, Akos; Guttman, A.

In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 1032, 01.10.2016, p. 139-143.

Research output: Contribution to journalArticle

Szigeti, Marton ; Bondar, Judit ; Gjerde, Douglas ; Keresztessy, Zsolt ; Szekrenyes, Akos ; Guttman, A. / Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns. In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 2016 ; Vol. 1032. pp. 139-143.
@article{4e2eb09659db4873ae5091a9e0c4adb1,
title = "Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns",
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 10 min 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.",
keywords = "Capillary electrophoresis, Enzyme immobilization, N-glycosylation, PNGase F",
author = "Marton Szigeti and Judit Bondar and Douglas Gjerde and Zsolt Keresztessy and Akos Szekrenyes and A. Guttman",
year = "2016",
month = "10",
day = "1",
doi = "10.1016/j.jchromb.2016.02.006",
language = "English",
volume = "1032",
pages = "139--143",
journal = "Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences",
issn = "1570-0232",
publisher = "Elsevier",

}

TY - JOUR

T1 - Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns

AU - Szigeti, Marton

AU - Bondar, Judit

AU - Gjerde, Douglas

AU - Keresztessy, Zsolt

AU - Szekrenyes, Akos

AU - Guttman, A.

PY - 2016/10/1

Y1 - 2016/10/1

N2 - 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 10 min 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.

AB - 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 10 min 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.

KW - Capillary electrophoresis

KW - Enzyme immobilization

KW - N-glycosylation

KW - PNGase F

UR - http://www.scopus.com/inward/record.url?scp=85027917216&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85027917216&partnerID=8YFLogxK

U2 - 10.1016/j.jchromb.2016.02.006

DO - 10.1016/j.jchromb.2016.02.006

M3 - Article

C2 - 26899450

AN - SCOPUS:85027917216

VL - 1032

SP - 139

EP - 143

JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

SN - 1570-0232

ER -