title: Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues.
abstract: Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.
authors.affiliation: Analytical Chemistry, Genentech Inc., One DNA Way, South San Francisco, California 94080, USA. sraju@gene.com
authors.forename: T S
authors.initials: TS
authors.lastname: Raju
authors.forename: J B
authors.initials: JB
authors.lastname: Briggs
authors.forename: S M
authors.initials: SM
authors.lastname: Chamow
authors.forename: M E
authors.initials: ME
authors.lastname: Winkler
authors.forename: A J
authors.initials: AJ
authors.lastname: Jones
mesh_terms: D000117:Acetylglucosamine / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000188:drug therapy; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D006224:Cricetinae; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D005690:Galactose / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism* / Q000627:therapeutic use; D006031:Glycosylation; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D007142:Immunoglobulin gamma-Chains; D015636:Magnesium Chloride / Q000378:metabolism; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D018805:Sepsis / Q000188:drug therapy; D012799:Sialyltransferases / Q000378:metabolism; D014162:Transfection; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D006023:Glycoproteins; D007143:Immunoglobulin Heavy Chains; D007142:Immunoglobulin gamma-Chains; D009844:Oligosaccharides; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D015636:Magnesium Chloride; D014531:Uridine Diphosphate Galactose; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; C099374:Ro 45-2081; D009237:N-Acetyllactosamine Synthase; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine; D005690:Galactose; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase