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Glycobiology Advance Access published online on May 22, 2006
Glycobiology, doi:10.1093/glycob/cwl004
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© 2006 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited
Received February 8, 2006
Revised May 11, 2006
Accepted May 13, 2006

Article

GlycoPegylation of recombinant therapeutic proteins produced in E. coli

Shawn DeFrees 1, Zhi-Guang Wang 1, Ruye Xing 1, Arthur E. Scott 1, Jin Wang 1, David Zopf 1 *, Dominique L. Gouty 2, Eric R. Sjoberg 2, Krishnasamy Panneerselvam 2, Els C.M. Brinkman-Van der Linden 3, Robert J. Bayer 2, Mads A. Tarp 4, and Henrik Clausen 4

1 Neose Technologies, Inc. 102 Witmer Road Dr. Horsham, PA 199044
2 Neose Technologies, Inc. 6330 Nancy Ridge Road, Suites 102-103 San Diego, CA 92121
3 Neose Technologies, Inc. 6330 Nancy Ridge Road, Suites 102-103 San Diego, CA 92121; Current Addresses: Els C.M. Brinkman-Van der Linden Noordwijkerhout 2211 AR Schaepmanlaan 19, The Netherlands
4 Department of Medical Biochemistry and Genetics Glycobiology University of Copenhagen Blegdamsvej 3 DK-2200 N, Denmark

* To whom correspondence should be addressed.
David Zopf, E-mail: dzopf{at}neose.com


  Abstract

Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong half-life and enhance pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc-glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in E. coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, G-CSF, IFN-{alpha}2b, and GM-CSF, which are currently in clinical use.

Keywords: G-CSF/IFN-{alpha}2b/GM-CSF/pegylation/glycosylation.
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