Next Article in Journal
Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease
Next Article in Special Issue
Biologics in Dermatology
Previous Article in Journal / Special Issue
Inactivation of Caliciviruses
Pharmaceuticals 2013, 6(3), 393-406; doi:10.3390/ph6030393
Article

Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

,
 and *
Vaccine Analytical Development, Merck Research Laboratories, West Point, PA 19486, USA Present Address: Sino Biological, Inc., 14 Zhong He St., BDA, Beijing 100176, China.
* Author to whom correspondence should be addressed.
Received: 29 January 2013 / Accepted: 13 March 2013 / Published: 21 March 2013
(This article belongs to the Special Issue Biologics)
View Full-Text   |   Download PDF [697 KB, 22 March 2013; original version 21 March 2013]   |   Browse Figures

Abstract

The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis.
Keywords: CE-LIF; APTS; HILIC-HPLC; glycans; monoclonal antibody NS0; capillary electrophoresis CE-LIF; APTS; HILIC-HPLC; glycans; monoclonal antibody NS0; capillary electrophoresis
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Share & Cite This Article

Export to BibTeX |
EndNote


MDPI and ACS Style

Hamm, M.; Wang, Y.; Rustandi, R.R. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection. Pharmaceuticals 2013, 6, 393-406.

View more citation formats

Related Articles

Article Metrics

Comments

Citing Articles

[Return to top]
Pharmaceuticals EISSN 1424-8247 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert