title: Design, synthesis, and assessment of a de novo affinity adsorbent for the purification of recombinant human erythropoietin.
abstract: This work describes the assessment of a de novo synthetic affinity ligand for recombinant human erythropoietin (rHuEPO), based on the multicomponent Ugi reaction. Four Ugi ligands were designed based on the X-ray crystallographic structure of the complex between human erythropoietin and site 1 of its cell-surface receptor (EPObp)2 ; screening of the ligands with pure rHuEPO samples identified a lead ligand (A9C10I8) immobilized on aldehyde-functionalized agarose beads, which was able to bind and elute erythropoietin, as determined by SDS-PAGE and Western blot analyses. Furthermore, small-scale affinity chromatography performed on the immobilized adsorbent showed its ability to isolate rHuEPO from a spiked mammalian cell supernatant with a purity of ∼80%, as estimated with gel densitometry. This approach could lead to the development of a cost-effective downstream process for rHuEPO, as an alternative to the current multi-step purification protocols.
authors.affiliation: Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, United Kingdom. ge235@cam.ac.uk.
authors.forename: Graziella
authors.initials: G
authors.lastname: El Khoury
authors.forename: Yian
authors.initials: Y
authors.lastname: Wang
authors.forename: Di
authors.initials: D
authors.lastname: Wang
authors.forename: Shaleem I
authors.initials: SI
authors.lastname: Jacob
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Lowe
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D018360:Crystallography, X-Ray; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C554270:EPO protein, human; D011994:Recombinant Proteins; D004921:Erythropoietin
keywords: Ugi multicomponent reaction; affinity chromatography; purification of rHuEPO; synthetic affinity adsorbent