Purification of Oligonucleotides by High Affinity, Low Molecular Weight Displacers

Abstract

High affinity, low molecular weight anionic displacers were successfully employed for the purification of antisense oligonucleotides. Several important structural characteristics were identified that contribute to the affinity of low molecular weight displacers to a hydrophilized polystyrene divinyl benzene anion exchanger. Sulfonic acid groups were found to possess higher affinity than carboxylic acid and phosphate functionalities, and nonspecific interactions (particularly hydrophobic interactions) were shown to play a major role in the retention process on this stationary phase material. Using this information, two high affinity, low molecular weight displacers were identified. These molecules are relatively inexpensive organic dyes that possess multiple sulfonic acid moieties, as well as aromatic functionalities, which increase nonspecific interactions with the stationary phase. These high affinity displacers, which can be readily detected, were then employed to displace several strongly retained antisense oligonucleotides that could not be displaced by previously established low molecular weight displacers. The displacement process resulted in very high purities of the antisense oligonucleotides. The results presented in this paper are significant in that they demonstrate that low molecular weight displacers for ion-exchange chromatography can possess equal to or greater affinities than their higher molecular weight counterparts, when nonspecific interactions with the stationary phase are exploited. In addition, the results illustrate the high resolutions possible with displacement chromatography and demonstrate an attractive technology for the process scale purification of oligonucleotides.

Unique selectivity windows using selective displacers/eluents and mobile phase modifiers on hydroxyapatite

“…A Jupiter 5 m C4 300A column (4.6 mm × 50 mm) was purchased from Phenomenex (Torrance, CA). Ribonuclease A from bovine pancreas (RNaseA), ribonuclease B from bovine pancreas (RNaseB), ␣-chymotrypsinogen A from bovine pancreas (␣-ChyA), cytochrome C from equine heart (CytC), lysozyme from chicken egg white (Lys), conalbumin from chicken egg white (Conal), hemoglobin from bovine blood (Hemo), myoglobin from equine heart (Myo), avidin from chicken egg white, subtilisin A from Bacillus, elastase from porcine pancreas, papain from papaya latex, bromelain from pineapple stem, alcohol dehydrogenase from equine liver, trypsinogen from bovine pancreas, catalase from bovine liver, aprotinin from bovine lung, aconitase from porcine heart, albumin from bovine serum, neomycin sulfate (displacer 1), paromomycin sulfate (2), bekanamycin sulfate (3), amikacin sulfate (4), spermine (5), bis(hexamethylene)triamine (7), spermidine (8), 1,4-bis(3-aminopropyl)piperazine (9), diethylenetriamine (10), 4,7,10-trioxa-1,13-tridecanediamine (11), N,Ndiethyl-1,3-propanediamine (12), N,N-diethyldiethylenetriamine (13), 2-(2-aminoethylamino)ethanol (14), spectinomycin dihydrochloride pentahydrate (15), l-arginine methyl ester dihydrochloride (16), l-lysine methyl ester dihydrochloride (17), N-hexylethylenediamine (18), piperazine (19), cyclohexylamine (20), acetic acid (21), malonic acid (22), succinic acid (23), adipic acid (24), isocitric acid lactone (25), trans-aconitic acid (26), 1,2,4-butanetricarboxylic acid (27), 1,2,3,4-butanetetracarboxylic acid (28), glycine (29), 3-guanidinopropionic acid (30), 5-aminovaleric acid (31), pantothenic acid (32), aspartic acid (33), l-␤-homoglutamic acid hydrochloride (34), guanidinosuccinic acid (35), l-2,3-diaminopropionic acid hydrochloride (36), lysine (37), arginine (38), meso-2,3,-diaminosuccinic acid (39), ethylenediaminetetrapropionic acid (40), glycerol (41), threitol (42), adonitol (43), dulcitol (44), malic acid (45), tartaric acid (46), mucic acid …”

“…Displacement chromatography has been successfully employed for the purification of proteins on multiple different stationary phases [2][3][4][5][6][7][8][9][10][11][12][13]. A wide variety of classes of displacers, such as polyelectrolytes [9], polysacchardies [14], low-molecular-mass dendrimers [11], amino acids [15], antibiotics [16] and aminoglycosidepolyamines [17] have been identified for protein displacement separations.…”

Characterization and design of chemically selective cationic displacers using a robotic high‐throughput screen

“…Displacement chromatography has been successfully employed for the purification of proteins using hydroxyapatite,1, 2 hydrophobic interaction,3 and ion exchange chromatographic systems 4–14. In particular, ion exchange displacement chromatography has attracted significant attention as a powerful technique for the purification of biomolecules 7, 8, 15.…”

Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing

“…Notwithstanding the reduced environmental impact of employing aqueous-based purifications, there is room to improve the chromatographic techniques employed. AEX chromatography in displacement mode was suggested several years ago. − However, the development was performed in an era when full understanding of the various process impurities was not available, and no further efforts have been made since. Displacement chromatography can be very efficient and generate concentrated, purified product, potentially with significant reduction in solvent usage, so a revisit may be warranted.…”