title: A carbohydrate-binding affinity ligand for the specific enrichment of glycoproteins.
abstract: One challenge facing the production of glycoprotein therapeutics is the lack of stable and selective affinity ligands for their enrichment. Synthetic affinity ligands based on the solid phase multi-component Ugi reaction represent a desirable option, particularly those incorporating benzoboroxole and its derivatives, which have been shown to enrich glycoproteins under physiological conditions. Thus, an Ugi ligand, A21C11I8, comprising 5-amino-2-hydroxymethylphenylboronic acid was synthesised on aldehyde-functionalised Sepharose™. Immobilised A21C11I8 displayed affinity for the glycosylated protein, glucose oxidase (GOx), which bound primarily through its glycan moiety. The ligand had a preference for sugar alcohols and the furanose form of the monosaccharides tested. Compared with immobilised 3-aminophenylboronic acid and Concanavalin A, the Ugi ligand was able to purify GOx from spiked Escherichia coli supernatants with retention of its maximum enzymatic activity and protein recovery. Glycan profiles of human immunoglobulin G tested on A21C11I8 columns suggested that the adsorbent possesses the potential to resolve sialylated and neutral glycoforms. The benzoboroxole-functionalised Ugi ligand may find application in selective glycoform separation.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK; Bioprocessing Technology Institute, A*STAR-Agency for Science, Technology and Research, Singapore.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK.
authors.forename: Graziella
authors.initials: G
authors.lastname: El Khoury
authors.affiliation: Bioprocessing Technology Institute, A*STAR-Agency for Science, Technology and Research, Singapore; Department of Anatomy, National University of Singapore, Singapore.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Bioprocessing Technology Institute, A*STAR-Agency for Science, Technology and Research, Singapore; GlycoScience Group, NIBRT-The National Institute for Bioprocessing Research and Training, Mount Merrion, Blackrock, Co., Dublin, Ireland.
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK. Electronic address: chen_chen@bti.a-star.edu.sg.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Lowe
mesh_terms: D000327:Adsorption; D001897:Boronic Acids / Q000737:chemistry / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008024:Ligands; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism
chemical_list: D001897:Boronic Acids; D006023:Glycoproteins; D007074:Immunoglobulin G; D008024:Ligands; D009005:Monosaccharides; C028592:3-aminobenzeneboronic acid; D005949:Glucose Oxidase
keywords: Affinity chromatography; Benzoboroxole; Glycoform selection; Glycoprotein purification