title: Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids.
abstract: Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble β-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 μM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz.
authors.affiliation: Chemistry Department, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.
authors.forename: Edward
authors.initials: E
authors.lastname: Bodnar
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, H4P 2R2, Canada.|Département de biochimie et médecine moléculaire, Université de Montréal, Montreal, QC, H3C 3J7, Canada.
authors.forename: Céline
authors.initials: C
authors.lastname: Raymond
authors.affiliation: Chemistry Department, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Lopez
authors.affiliation: Microbiology Department, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.
authors.forename: Carina
authors.initials: C
authors.lastname: Villacrés
authors.affiliation: Microbiology Department, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Butler
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, K1A 0R6, Canada.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Schoenhofen
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Montreal, QC, H4P 2R2, Canada.|Département de biochimie et médecine moléculaire, Université de Montréal, Montreal, QC, H3C 3J7, Canada.
authors.forename: Yves
authors.initials: Y
authors.lastname: Durocher
authors.affiliation: Chemistry Department, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada. Helene.Perreault@umanitoba.ca.
authors.forename: Hélène
authors.initials: H
authors.lastname: Perreault
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D060847:Metabolic Engineering; D015394:Molecular Structure; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012794:Sialic Acids / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D011134:Polysaccharides; D012794:Sialic Acids; D009237:N-Acetyllactosamine Synthase
keywords: Azido-sialic acid; Enzymatic sialylation; MALDI-ToF-MS; Metabolic sialylation; Monoclonal antibodies; Sialylation