Purification of an oligonucleotide at high column loading by high affinity, low-molecular-mass displacers.
The development of efficient techniques for large-scale oligonucleotide purification is of great interest due to the increased demand for antisense oligonucleotides as therapeutics as well as their use for target validation and gene functionalization. This paper describes the use of anion-exchange displacement chromatography for the purification of 20-mer phosphorothioate oligonucleotide from its closely related impurities using low-molecular-mass amaranth as the displacer. Experiments were carried out to examine the effect of the feed load on the performance of the displacement chromatography. In contrast to prior work, displacement chromatography was successfully scaled-up to high column loadings while maintaining high purity and yields. Experiments carried out on a Source 15Q column indicated that crude oligonucleotide loading as high as 39.2 mg/ml of column were readily processed, resulting in product recovery of 86% and purity of 92%. These results demonstrate that anion-exchange displacement chromatography can indeed be employed for large-scale oligonucleotide separations at high column loading.
Tugcu
N
N
Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA.
Deshmukh
R R
RR
Sanghvic
Y S
YS
Moored
J A
JA
Cramer
S M
SM
D000548:Amaranth Dye; D000838:Anions; D002852:Chromatography, Ion Exchange / Q000379:methods*; D009841:Oligonucleotides / Q000302:isolation & purification*
D000838:Anions; D009841:Oligonucleotides; D000548:Amaranth Dye