title: High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation.
abstract: Recent advances in miniaturized cell culture systems have facilitated the screening of media additives on productivity and protein quality attributes of mammalian cell cultures. However, intracellular components are not routinely measured due to the limited throughput of available analytical techniques. In this work, time profiling of intracellular nucleotides and nucleotide sugars of CHO-S cell fed-batch processes in a micro-scale bioreactor system was carried out using a recently developed high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS). Supplementation of various media additives significantly altered the intracellular nucleotides and nucleotide sugars that are inextricably linked to the process of glycosylation. The results revealed that UDP-Gal synthesis appeared to be particularly limiting whereas the impact of elevated UDP-GlcNAc and GDP-Fuc levels on the final glycosylation patterns was only marginally important. In contrast, manganese and asparagine supplementation altered the glycan profiles without affecting intracellular components. The combination of miniaturized cell cultures and high-throughput analytical techniques serves therefore as a useful tool for future quality driven media optimization studies.
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, CH- 8093 Zurich, Switzerland.
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Villiger
authors.affiliation: Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Steinhoff
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, CH- 8093 Zurich, Switzerland.
authors.forename: Marija
authors.initials: M
authors.lastname: Ivarsson
authors.affiliation: Merck Serono SA, Corsier-sur-Vevey, Biotech Process Sciences, ZI B, CH-1809 Fenil-sur-Corsier, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Solacroup
authors.affiliation: Merck Serono SA, Corsier-sur-Vevey, Biotech Process Sciences, ZI B, CH-1809 Fenil-sur-Corsier, Switzerland.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Stettler
authors.affiliation: Merck Serono SA, Corsier-sur-Vevey, Biotech Process Sciences, ZI B, CH-1809 Fenil-sur-Corsier, Switzerland.
authors.forename: Hervé
authors.initials: H
authors.lastname: Broly
authors.affiliation: Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Krismer
authors.affiliation: Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.affiliation: Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, CH- 8093 Zurich, Switzerland.
authors.forename: Massimo
authors.initials: M
authors.lastname: Morbidelli
authors.affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, CH- 8093 Zurich, Switzerland; Department of Chemical Engineering, University of Chemistry and Technology, Technicka 5, 166 28 Prague, Czech Republic. Electronic address: miroslav.soos@chem.ethz.ch.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Soos
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D009711:Nucleotides / Q000032:analysis* / Q000737:chemistry*
chemical_list: D000906:Antibodies; D009711:Nucleotides
keywords: High-throughput; MALDI-TOF-MS; Media optimization; Micro-scale bioreactors; N-linked glycosylation; Nucleotide sugars