title: Purification of a highly modified RNA-aptamer. Effect of complete denaturation during chromatography on product recovery and specific activity.
abstract: To evaluate RNA-aptamers as potential drug candidates, efficient and scaleable purification protocols are needed. Because aptamers are highly structured and rigid molecules, denaturation during the purification process is a critical aspect to obtain a pure and active product. A two-step chromatographic procedure was developed to purify a synthetic anti-VEGF aptamer at the preparative scale. A reversed-phase chromatographic step was optimized with a highly hydrophobic ion pairing reagent, followed by ion-exchange chromatography in which heat and a chaotropic salt were used. Because of the presence of 2'-modified ribose, denaturation conditions had to be optimized in both chromatographic steps to achieve a fully active molecule.
authors.affiliation: NeXstar Pharmaceuticals Inc., Boulder, CO 80301, USA. pbridonneau@nexstar.com
authors.forename: P
authors.initials: P
authors.lastname: Bridonneau
authors.forename: S
authors.initials: S
authors.lastname: Bunch
authors.forename: R
authors.initials: R
authors.lastname: Tengler
authors.forename: K
authors.initials: K
authors.lastname: Hill
authors.forename: J
authors.initials: J
authors.lastname: Carter
authors.forename: W
authors.initials: W
authors.lastname: Pieken
authors.forename: D
authors.initials: D
authors.lastname: Tinnermeier
authors.forename: R
authors.initials: R
authors.lastname: Lehrman
authors.forename: D W
authors.initials: DW
authors.lastname: Drolet
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D016228:Endothelial Growth Factors / Q000235:genetics; D008222:Lymphokines / Q000235:genetics; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D016228:Endothelial Growth Factors; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012313:RNA