title: An optimized method for extraction and quantification of nucleotides and nucleotide sugars from mammalian cells.
abstract: Glycosylation is a critical attribute of therapeutic proteins given its impact on the clinical safety and efficacy of these molecules. The biochemical process of glycosylation is inextricably dependent on metabolism and ensuing availability of nucleotides and nucleotide sugars (NSs) during cell culture. Herein, we present a comprehensive methodology to extract and quantify these metabolites from cultured cells. To establish the full protocol, two methods for the extraction of these compounds were evaluated for efficiency, and the requirement for quenching and washing the sample was assessed. A chromatographic method based on anion exchange has been optimized to separate and quantify eight nucleotides and nine NSs in less than 30 min. Degradation of nucleotides and NSs under extraction conditions was evaluated to aid in selection of the most efficient extraction protocol. We conclude that the optimized chromatographic method is quick, robust, and sensitive for quantifying nucleotides and NSs. Furthermore, our results show that samples taken from cell culture should be treated with 50% v/v acetonitrile and do not require quenching or washing for reliable extraction of nucleotides and NSs. This comprehensive protocol should prove useful in determining the impact of nucleotide and NS metabolism on protein glycosylation.
authors.affiliation: Department of Chemical Engineering and Chemical Technology, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
authors.forename: Ioscani Jimenez
authors.initials: IJ
authors.lastname: del Val
authors.forename: Sarantos
authors.initials: S
authors.lastname: Kyriakopoulos
authors.forename: Karen M
authors.initials: KM
authors.lastname: Polizzi
authors.forename: Cleo
authors.initials: C
authors.lastname: Kontoravdi
mesh_terms: D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003412:Cricetulus; D006031:Glycosylation; D009711:Nucleotides / Q000302:isolation & purification*
chemical_list: D002241:Carbohydrates; D009711:Nucleotides
keywords: CHO cells; HPLC; Metabolite extraction; Nucleotide sugars; Nucleotides; Protein glycosylation