GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli

• Published in: Glycobiology (Oxford) 2006-09, Vol.16 (9), p.833-843
• Main Authors: DeFrees, Shawn, Wang, Zhi-Guang, Xing, Ruye, Scott, Arthur E, Wang, Jin, Zopf, David, Gouty, Dominique L, Sjoberg, Eric R, Panneerselvam, Krishnasamy, Brinkman-Van der Linden, Els C.M, Bayer, Robert J, Tarp, Mads A, Clausen, Henrik
• Format: Article
• Language: English
• Subjects: 2-(4-morpholino)-ethane sulfonic acid; Acetylgalactosamine - analogs & derivatives; Acetylgalactosamine - chemistry; area under the concentration–time curve; AUC; CMP-SiaPEG; cytidine monophosphate 5′-aminoglycyl-methoxypolyeneglycol neuraminic acid; Cytokines - biosynthesis; Cytokines - chemistry; Cytokines - therapeutic use; Escherichia coli; G-CSF; GalNAc-T; Glycosylation; GM-CSF; granulocyte colony stimulating factor; granulocyte/macrophage colony stimulating factor; high-performance liquid chromatography; HPLC; Humans; IFN-α2b; IgG; immunoglobulin G; interferon-alpha2b; LC-MS/MS; liquid chromatography-mass spectrometry/mass spectrometry; Madin–Darby bovine kidney; MALDI-TOF/MS; matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; MDBK; MES; PEG; PEGylation; polyethylene glycol; Polyethylene Glycols - chemistry; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - therapeutic use; SDS–PAGE; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; UDP-GalNAc: polypeptide α-N-acetylgalactosaminyltransferase; vesicular stomatitis virus; VSV; WBC; white blood cell
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwl004

Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the 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 Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-α2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use.