Purification of a highly modified RNA-aptamer. Effect of complete denaturation during chromatography on product recovery and specific activity.
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.
Bridonneau
P
P
NeXstar Pharmaceuticals Inc., Boulder, CO 80301, USA. pbridonneau@nexstar.com
Bunch
S
S
Tengler
R
R
Hill
K
K
Carter
J
J
Pieken
W
W
Tinnermeier
D
D
Lehrman
R
R
Drolet
D W
DW
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
D016228:Endothelial Growth Factors; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012313:RNA