Chromatographic Assessment of Oligonucleotide...
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ABSTRACT The need for multiple approaches to determine API yields and iden- tify contaminants generated during therapeutic oligonucleotide (ON) development stems from regulatory guidelines. Among the possible contaminants unique to RNA are linkage isomers that are difficult to identify by MS and LC-MS techniques. Using non-porous bead-based and higher capacity monolithic anion exchange phases for ON analysis and purification, we demonstrate an alternative method for evaluation of these isomers, and suggest an independent linkage-confirming tech- nique. Non-porous bead and monolithic columns support resolution of ONs with control of selectivity, and improved sample throughput in R&D or quality assessment environments. The monolithic phases are scalable to laboratory scale purification. We will discuss specific approaches for evaluation of contaminants unique to synthetic RNA, as well as options for scalable purification. Oligonucleotide Linkage vs Retention: DNAPac PA200 4 × 250mm Column RNA synthetic routes may produce occasional 2´– 5´ phosphodiester bonds where biologically ‘normal” RNA would have 3´– 5´ linkages. During the TIDES 2005 conference the opinion was expressed that the presence of these aberrant linkages are likely to have biological conse- quences, and that they represent a difficult analytical challenge because: 1. They do not introduce a change in mass that might be detected by mass spectrometry 2. They are not expected to introduce a change in hydrophobicity that would produce a selectivity change in reversed-phase chromatography, and 3. They are unlikely to produce changes in ionic character of RNA that would allow differentiation by ion-exchange chromatography. TIDES 2006 Presentation Chromatographic Assessment of Oligonucleotide Purity J.R. Thayer, S. Rao, and S. Xie, Dionex Corporation, Sunnyvale, CA, USA; N. Puri and C. Burnett, Ambion Inc., Austin, TX, USA Chromatographic Assessment of Oligonucleotide Purity J.R. Thayer, S. Rao, and S. Xie, Dionex Corporation, Sunnyvale, CA, USA; N. Puri and C. Burnett, Ambion Inc., Austin, TX, USA Initial studies employed poly-A or poly-U 21 mers with 0, 2, 4, or 8 consecutive 2´– 5´ linkages at the 5’ end of the oligonucleotide. Figure 1 shows five consecutive 2´– 5´ linkages. Figure 1. Ribonucleic Acid (RNA) Linkage Isomers 2´– 5´ Linkages (aberrant) 3´– 5´ Linkages (Normal) 5´ 3´ HO OH O O Base OH O O Base O P OH O O Base O P OH O O Base O P O O O O O O O O O O OH O O Base O P O P O O O H O O Base O P O O OH O OH Base O P OH O O Base O P O O O H O O Base O P O O O H O O Base O P O O OH O O Base In Figure 2 a curved NaCl gradient applied to the DNAPac PA200 column readily resolves the cartridge-purified rA 21 mer RNAs in order of decreasing number of 2´– 5´ linkages. 0 22 0 200 rA 21 -L0 rA 21 -L2 rA 21 -L4 rA 21 -L8 15.17 16.21 17.06 18.40 Flow: 1.20 mL/min 1.25M NaCl: 10.0 % 40.0 70.0 10.0 mA Minutes Figure 2. Resolution of Linkage Isomers of rA 21 Oligonucleotides: Oligos with 0, 2, 4 and 8, 2’–5’ Linkages on 5’ End, 30 °C, pH 8
Transcript of Chromatographic Assessment of Oligonucleotide...
TIDES2006Presentation �
ABSTRACTThe need for multiple approaches to determine API yields and iden-tify contaminants generated during therapeutic oligonucleotide (ON) development stems from regulatory guidelines. Among the possible contaminants unique to RNA are linkage isomers that are difficult to identify by MS and LC-MS techniques. Using non-porous bead-based and higher capacity monolithic anion exchange phases for ON analysis and purification, we demonstrate an alternative method for evaluation of these isomers, and suggest an independent linkage-confirming tech-nique. Non-porous bead and monolithic columns support resolution of ONs with control of selectivity, and improved sample throughput in R&D or quality assessment environments. The monolithic phases are scalable to laboratory scale purification. We will discuss specific approaches for evaluation of contaminants unique to synthetic RNA, as well as options for scalable purification.
Oligonucleotide Linkage vs Retention: DNAPac PA200 4 × 250mm ColumnRNA synthetic routes may produce occasional 2´– 5´ phosphodiester bonds where biologically ‘normal” RNA would have 3´– 5´ linkages. During the TIDES 2005 conference the opinion was expressed that the presence of these aberrant linkages are likely to have biological conse-quences, and that they represent a difficult analytical challenge because:
1. They do not introduce a change in mass that might be detected by mass spectrometry
2. They are not expected to introduce a change in hydrophobicity that would produce a selectivity change in reversed-phase chromatography, and
3. They are unlikely to produce changes in ionic character of RNA that would allow differentiation by ion-exchange chromatography.
TIDES2006Presentation
Chromatographic Assessment of Oligonucleotide PurityJ.R. Thayer, S. Rao, and S. Xie, Dionex Corporation, Sunnyvale, CA, USA; N. Puri and C. Burnett, Ambion Inc., Austin, TX, USA
Chromatographic Assessment of Oligonucleotide PurityJ.R. Thayer, S. Rao, and S. Xie, Dionex Corporation, Sunnyvale, CA, USA; N. Puri and C. Burnett, Ambion Inc., Austin, TX, USA
Initial studies employed poly-A or poly-U 21 mers with 0, 2, 4, or 8 consecutive 2´– 5´ linkages at the 5’ end of the oligonucleotide. Figure 1 shows five consecutive 2´– 5´ linkages.
Figure 1. Ribonucleic Acid (RNA) Linkage Isomers
2´– 5´ Linkages(aberrant)
3´– 5´ Linkages (Normal)
5´
3´
HO
OH
O
O
Base
OH
O
O
Base
OP
OH
O
O
Base
OPOH
O
O
Base
OP
O OO O
O OO O
O O
OH
O
O
Base
OP
OP
OO
OH
O
O
Base
OPOO
O H
O
O H
Base
OP OH
O
O
Base
OP
O OOH
O
O
Base
OP
O OOH
O
O
Base
OP
OO
O H
O
O
Base
In Figure 2 a curved NaCl gradient applied to the DNAPac PA200 column readily resolves the cartridge-purified rA21 mer RNAs in order of decreasing number of 2´– 5´ linkages.
0 220
200
rA21-L0
rA21-L2
rA21-L4
rA21-L8
15.1
7
16.2
1
17.0
6
18.4
0
Flow: 1.20 mL/min
1.25M NaCl: 10.0 %
40.0
70.0
10.0
mA
Minutes
Figure 2. Resolution of Linkage Isomers of rA21 Oligonucleotides:Oligos with 0, 2, 4 and 8, 2’–5’ Linkages on 5’ End, 30 °C, pH 8
Anuta
New Stamp
2 ChromatographicAssessmentofOligonucleotidePurity
In Figure 3 the curved NaCl gradient applied to the DNAPac PA200 column readily resolves the machine-grade rU21 mer RNAs in order of decreasing number of 2´– 5´ linkages. Since elution before the normal full length oligomer is characteristic of failure sequences often contami-nating purified oligonucleotides, an independent assay is necessary to confirm the presence of the aberrant linkages.
The exonucleases in Figure 4 excise terminal monophosphates from oligonucleotides. Phosphodiesterase-I (PDAse-I) processes from the 3´ end, producing 5´ monophosphates, and Phosphodiesterase-II (PDAse-II) processes from the 5´ end, producing 3´ monophosphates.
If one or both of these exonucleases fails to process through 2´– 5´ linkages, it should produce oligonucleotide fragments indicating the presence of those aberrant linkages.
In Figure 5 the PDAse-II control shows no interfering peaks and the undigested rA21 mer elutes at ~ 19 minutes.
Exposure of the rA21mer to either PDAse reveals complete digestion by both enzymes in 18 hr.
In Figure 6 the PDAse-I control shows no interfering peaks and the undigested rA21 mers with 0-, 2, 4, and 8 aberrant linkages elute between 15.1 and 18.5 minutes.
Exposure of these compounds to PDAse-I reveals complete digestion of all oligonucleotides in 18 hr.
0 200
90
rU21L0
rU21L2
rU21L4
rU21L8
15.9
2
16.8
717
.41
17.9
1Flow: 1.20 mL/min
1.25M NaCl:
10.0 %
40.0
70.0
10.0
mA 26
0
Minutes
Figure 3. Resolution of Linkage Isomers of rU21 Oligonucleotides:Crude Oligo with 0, 2, 4 and 8, 2’-5’ linkages on 5’ end, 30°C, pH 8 Curve 3
16124 80 24
0
250
PDAse-II, Ctrl
rA21
rA21PDAse-I, 18 hr
rA21PDAse-II, 18 hr
19.3
5
Flow: 1.20 mL/min
1.25M NaCl:
10.0 %
40.0
70.0
4.0
mA2
60
Minutes
Figure 5. Assay of PDAse-I, -II Digestion:18 hr Exposure at 37 °C rA21 Digest Controls, 30°C, pH8, 50 – 500 mM in 20 min, Curve 3
12 20164 8
0
200
PDAse-I, Ctrl
rA21L0
rA21L2
rA21L4
rA21L8
rA21L0 PDAse-I
rA21L2 PDAse-I
rA21L4 PDAse-I
rA21L8 PDAse-I
Flow: 1.20 mL/min
1.25M NaCl:
10.0 %
40.0
70.0
10.0
18.4
0
17.0
6
16.2
1
15.1
7
Figure 6. Assay of ssRNA Digestion by Linkage: 18hrCartridge Purified rA21 Controls, 30°C, pH 8, 50–500 mM/20 min, Curve 3
0 24
mA 26
0
Minutes12 20164 8
Phosphodiesterase-IProduces 5´ NMPs
Phosphodiesterase-IIProduces 3´ NMPs
Figure 4. RNA 3´– 5´ vs 2´– 5´ Linkage: Exonuclease Assay
OP
O O
O HO
O H
OP
O O
OHO
OH O
OHO
O
OO
Base
Base
Base
Base
Base
Base
Base
OP
O O O H
OP
O O
OHO
OOP
O O
OHO
OOP
O O
OHO
O
( - - - - Cleavage sites)
3´
5´
TIDES2006Presentation �
In Figure 7 the undigested rA21 mers with 2 aberrant linkages elutes from the DNAPac PA200 column at 18.2 minutes, and exposure of this oligo to PDAse-I results in complete digestion.
Exposure of this compound to PDAse-II for 18 hours reveals a major digestion product at ~ 3.57 minutes, and minor products at 4.4, 5.2, 6.0, and 6.8 minutes. Exposure for more time (24 hr), and an additional 17 hours after addition of more enzyme did not change this pattern.
Undigested rA21 mers with no aberrant linkages elutes at 19.4 minutes, and exposure of this oligo to PDAse-II results in complete digestion (Figure 8). Exposure of this compound with 2, 4, or 8 aberrant linkages to PDAse-II for 41 hours reveals major digestion products at ~ 3.56 (rA21L2), 4.86 (rA21L4), and 7.38 (rA21L8) minutes. The DNAPac PA200 elution position of each of these fully digested products is consistent with oligos of length equal to the number of consecutive aberrant linkages.
Next (Figure 9) we repeated the assay with the pyrimidine base (U) to compare these results with the purine base (A). The results obtained with the rA21 mer are fully repeated with the rU21 mer. Hence, the PDAse-II product appears to work equally well for purines and pyrimidines.
RNA with inadvertent 2´– 5´ linkages (Figure 10 A) are unlikely to have them placed consecutively, or at multiple locations like the test oligo-nucleotides previously described.
However, they are likely to have only one inadvertent 2´– 5´ linkage, or if more than one, placed at non-adjacent positions, as suggested in Figure 10 B.
rA21L2
rA21L2 PDAse-I, 18 hr
rA21L2 PDAse-II, 18 hr
rA21L2 PDAse-II-2, 24 hr
rA21L2 PDAse-II-3, 41 hr3.56
4.40
5.19
6.00
6.83
3.57 4.41 5.20
6.01
6.83
3.58
4.41 5.19
5.99
6.82
18.2
0
Flow: 1.20 mL/min
1.25M NaCl:
10.0 %
40.0
70.0
10.0
Figure 7. Assay of ssRNA Digestion by Linkage: 18, 24, 41 hrrA21L2 Digest, 30°C, pH 8, 50–500 mM in 20 min, Curve 3
0 24Minutes
12 20164 80
200
mA 26
0
0 240
160
6.29
6.77
7.38
3.58
4.23
4.86
3.56
4.40
5.19
6.00
6.83 19
.38
Flow: 1.20 mL/min
1.25M
NaCl:4.0 %
40.0
70.0
4.0
mA 26
0
Minutes
Figure 8. Assay of ssRNA Digestion by Linkage: 41hrrA21LX Digest, 30°C, pH 8, 50–500 mM in 20 min, Curve 3
rA21L8 PDAse-II, 41 hr
rA21L4 PDAse-II, 41 hr
rA21L2 PDAse-II, 41 hr
rA21L0 PDAse-II 41 hr
PDAse-II, Ctrl
rA21
rU21
rU21 L0 PDAse-II-3, 41 hr
rU21L2 PDAse-II-3, 41 hr
rU21L4 PDAse-II-3, 41 hr
rU21L8 PDAse-II-3, 41 hr
7.64
8.48
4.99
5.77
3.64
4.41
18.9
3
Flow: 1.20 mL/min
1.25M NaCl: 4.0 %
40.0
70.0
4.0
Figure 9. Assay of ssRNA Digestion by Linkage: 41hrrU21LX Digest, 30°C, pH 8, 50–500 mM in 20 min, Curve 3
0 24Minutes
12 20164 80
200
mA 26
0
Figure 10. Ribonucleic Acid (RNA) Linkage Isomer Placement
A
B
OPO O
OHO
O
Base
OP
O O
O HO
O H
Base
OP
OO
O H
O
O
Base
OPO O
OHO
O
Base
OP
O OO H
OO
Base
OPO
O
OHO
O
Base
OH OPO O
OHO
O
Base
OPO O
OHO
O
Base
OPO O
OHO
O
Base
OP
O OO H
OO
Base
OP
O O O H
O
O
Base
OP
O O
OH
OO
Base
OH OP
O O O H
O
O
Base
OP
O O O H
O
O
Base
OP
O O O H
O
O
Base
OPO O
OHO
O
Base
OPO O
OHO
O
Base
OPO O
OHO
O
Base
OPO O
OHO
O
Base
OPO O
O HO
OH
Base5´
5´
3´
3´
� ChromatographicAssessmentofOligonucleotidePurity
In Figure 11 the new suite of mixed-base 21-mers with aberrant link-ages were chromatographed on the DNAPac PA200 at pH 7. At this pH samples Dio-5, Dio-6, and Dio-9 are completely resolved from all other samples, and are shown as red in the table. The pairs unresolved at this pH are shown as black.
In Figure 12 the fully resolved components are not shown, and the rest of the new suite of mixed-base 21-mers with aberrant linkages were chromatographed on the DNAPac PA200 at pH 9. Components resolved at pH 9 are now depicted in red in the table.
In Figure 13 the fully resolved components are not shown, and the rest of the new suite of mixed-base 21-mers with aberrant linkages were chromatographed on the DNAPac PA200 at pH 10. Components resolved at pH 10 are now depicted in red in the table.
2.5 10.0–10
390
mA 2
60
8.37
7.93
7.89
7.88
7.86
7.84
7.78
7.71
7.69
7.44
7.11
0.33M NaClO4:
23.4 %
38.4
Minutes
Dio-9:L1:C15-16
Dio-4:L2:A1-2,G3-4
Dio-1:L0
Dio-11:L2:C18-19,U20-21
Dio-10:L1:U20-21
Dio-8:L1:A5-6
Dio-12:L2:U19-21
Dio-3:L2:AU1-3
Dio-2:L1:A1-2
Dio-5:A10
Dio-6:L2:AG10-12
Figure 11. Retention of Mixed-Base 21-merswith 2´–5´ Linkages: pH 7
DNAPac PA200, 66-132 mM NaClO4/10 min, 1 mL/min, 30 °C
Dio-
2:
D i o - 1 : 5 ´ A * U * G * A A* C U U C A * G * GGUC * A G C * U * U * G 3 ´
Sample: UnresolvedDio-1: 4, 8, 10, 11Dio-2: 3,Dio-3: 2,Dio-4: 1, 10, 11Dio-5:Dio-6
Dio-
10:
Dio-
12:
Dio-
11:
Dio-
9:
Dio-
6:
Dio-
5:
Dio-
8:
Dio-
4:
Dio-
3:
Sample: UnresolvedDio-8: 1, 10, 11, 12Dio-9Dio-10: 1, 4, 8, 11Dio-11: 1, 4, 8, 10Dio-12: 3, 8
Figure 12. Retention of Mixed-Base 21 merswith 2´–5´ Linkages: pH 9
DNAPac PA200, 76–142 mM NaClO4/10 min, 1 mL/min, 30 °C
2.5 10.0–10
340
Dio-4:L2:A1-2,G3-4 7.86
Dio-8:L1:A5-6 7.84
Dio-11:L2:C18-19,U20-21 7.81
Dio-1:L0 7.76
Dio-10:L1:U20-21 7.74
Dio-12:L2:U19-21 7.66
Dio-2:L1:A1-2 7.48
Dio-3:L2:A1-2,U2-3 7.46
0.33M NaClO4:
26.4 %
41.4
Minutes
Dio-
2:
D i o - 1 : 5 ´ A * U * G * A A * C U U C A * G * G G U C * A G C *U* U * G 3 ´
Dio-
10:
Dio-
12:
Dio-
11:
Dio-
8:
Dio-
4:
Dio-
3:
mA 2
60
Sample: UnresolvedDio-1: 4, 8, 10, 11Dio-2: 3,Dio-3: 2,Dio-4: 1, 10, 11
Sample: UnresolvedDio-8: 1, 10, 11, 12Dio-10: 1, 4, 8, 11Dio-11: 1, 4, 8, 10Dio-12: 3, 8
Dio-8:L1:A5-6 8.76
Dio-1:L0 8.68
Dio-11:L2:C18-19,U20-21 8.64
Dio-10:L1:U20-21 8.62
Dio-2:L1:A1-2 8.58
Dio-3:L2:A1-2,U2-3 8.54
Dio-4:L2:A1-2,G3-4 8.53
0.33M NaClO4:
33.4 %
48.4
D i o - 1 : 5 ´ A * U * G * A A * C U U C A * G * G G U C * A G C * U * U * G 3 ´
Dio-
2:
Dio-
10:
Dio-
11:
Dio-
8:
Dio-
4:Di
o-3:
Figure 13. Retention of Mixed-Base 21 merswith 2´–5´ Linkages: pH 10
DNAPac PA200, 99–165 mM NaClO4/10 min, 1 mL/min, 30 °C
2.5 10.0Minutes–10
340
mA 2
60
Sample: UnresolvedDio-1: 10, 11Dio-2: 3,Dio-3: 2,Dio-4: 10, 11
Sample: UnresolvedDio-8: 11Dio-10: 1, 4, 11Dio-11: 1, 4, 8, 10
TIDES2006Presentation �
In Figure 14 the fully resolved components are not shown, and the rest of the new suite of mixed-base 21-mers with aberrant linkages were chromatographed on the DNAPac PA200 at pH 11. All components are now resolved at some pH and are now depicted in red in the table. Dio-2 and Dio-3 are not fully resolved, but can be differentiated at pH 11.
Since RNA is thought to degrade quickly at pH > 9, chromatography at high pH is usually discouraged. Here we observe that changes in flow (to increase the residence time of RNA on-column) during DNAPac PA200 chromatography at pH 11 does not change the relative amounts of the full-length sequence or impurities, indicating that exposure to pH 11 for 20 min or less does not result in RNA degradation on the DNAPac column.
Chromatography of PDAse-II treated mixed-base RNA 21 mers contain-ing one or two aberrant 2´– 5´ linkages at different positions in the RNA, result in specific, differentially retained, degradation products. The chromatographic elution position is indicative of the length of the digested fragment.
CONCLUSIONS: IObservations on RNA with aberrant 2´– 5´ linkages• Phosphodiesterase-I processes through RNA with 2´– 5´ linkages.• Phosphodiesterase-II appears to stop at 2´– 5´ linkages.• This Phosphodiesterase-II product appears to skip over 2´– 5´ link-
ages to produce partial digests from 5´-end located 2´– 5´ linkage oligos.
• Exhaustive (overnight) Phosphodiesterase-II treatment does not continue to process through 2´– 5´ linkages, even with extra enzyme and time.
• Exposure to pH 11 for 20 minutes or less does not reveal detectable on-column RNA degradation.
DNAPac PA200 • Resolves rA, and rU 21-mer 2´– 5´ linkage isomers.• Retains mixed-base RNA in a pH-dependent manner (as with DNA).• Resolves mixed-base 21-mer linkage isomers, but may require
different pH values to resolve all pairs.• Retains PDAse-II degradation products of each of the 11 mixed-base
2´– 5´ linkage isomers to unique positions.• Is recommended for analytical application at low sample
concentrations.
Dio-2:L1:A1-2
Dio-10:L1:U20
Dio-1:L0 7.74
7.66
7.62
Dio-11:L2:C18,U20 7.56
Dio-3:L2:A1-2,U2-3 7.55
0.33M NaClO4:40.4 %
55.4
Figure 14. Retention of Mixed-Base 21 merswith 2´–5´ Linkages: pH 11
DNAPac PA200, 122-188 mM NaClO4/10 min, 1 mL/min, 30 °C
2.5 10.0Minutes–10
340
mA 2
60
D i o - 1 : 5 ´ A * U * G * A A * C U U C A * G * G G U C * A G C * U * U * G 3 ´Di
o-2:
Dio-
10:
Dio-
11:
Dio-
3:
Sample: UnresolvedDio-1: 10, 11Dio-2: 3,Dio-3: 2,
Sample: UnresolvedDio-10: 1, 11Dio-11: 1, 10
1.25MNaCl:3.0 %
33.0
0 13.5–5
395
mA 2
60
1.44
1.53
1.60
2.47
2.93
3.36 4.05
4.25
4.47
4.16
4.83
5.55
5.36
5.98
1.42
1.60
5.68 6.34
12.8
4
1.65
1.53
Minutes
Figure 16. Elution of PDAse-II Digestion Products:DNAPac PA200, 10–119 mM NaClO4 /20 min, pH 8, 30 °C, 1 mL/min
Dio-1: 5´ AUGAACUUCAGGGUCAUCUUG 3 ´Dio-10: 5´ AUGAACUUCAGGGUCAUCUU*G 3 ´
Dio-12: 5´ AUGAACUUCAGGGUCAUCU*U*G 3 ´Dio-2: 5´ A*UGAACUUCAGGGUCAUCUUG 3 ´Dio-11: 5´ AUGAACUUCAGGGUCAUC*UU*G 3 ´
Dio-8: 5´ AUGAA*CUUCAGGGUCAUCUUG 3 ´Dio-6: 5´ AUGAACUUCA*G*GGUCAUCUUG 3 ´
Dio-5: 5´ AUGAACUUCA*GGGUCAUCUUG 3 ´Dio-9: 5´ AUGAACUUCAGGGUC*AUCUUG 3 ´
Dio-4: 5´ A*UG*AACUUCAGGGUCAUCUUG 3 ´Dio-3: 5´ A*U*GAACUUCAGGGUCAUCUUG 3 ´
Dio-1:L0
4.1 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.2–2
20Dio-1:L0
mAU
Minutes
A 0.93
0.63 1.25 1.89
95.3
0Flow: 1.00 ml/min
1.25M NaCl: 43.2 %59.6
6.15 7.00 8.00 9.00 10.00 11.00 12.15
Dio-1:L0
B 0.93
0.69 1.26 1.88
95.2
5Flow: 0.67 ml/min
1.25M NaCl: 44.3 %60.3
13.5 15.0 17.0 19.0 21.0 23.5
Dio-1:L0
Minutes
C 0.90
0.70 1.22 1.96
95.2
3
WVL: 260 nmFlow: 0.33 ml/min
1.25M NaCl: 46.9 %60.3
Figure 15. Effect of High pH on Stability of RNA: On-ColumnRelative Area (Area %) at 0.33 to 1 mL/min (pH 11)
WVL: 260 nm
WVL: 260 nm
–2
20
mAU
–2
20
mAU
Minutes
6 ChromatographicAssessmentofOligonucleotidePurity
EffECT Of LINkAgE ISOmERISm ON ThE PROSwIfT wAX-1S 4.6 × 50 mmMonolithic WAX retention
The ProSwift WAX Monolith
• Weak Anion exchange monolithic polymer phase.• Solid polymer rod with an uninterrupted interconnected network of
channels of controlled size range producing a morphology that sup-ports fast analyte mass transfer.
• Features high speed, low back-pressure separations with high resolution.
• The WAX monolith also exhibits 10–15 times the capacity of the pellicular DNAPac phase on a volume basis.
• These features support increased throughput and productivity.
0 7.5 15.0 22.50
4
2.25 µg
13.5 µg
120 µg
AU
Minutes
WVL:260 nmFlow: 1.00 ml/min
0
2.3
48 µg
432 µg
1.296 mg
AU
WVL:260 nm
DNAPac PA100 (4 × 50 mm)150–600 mM NaCl in 18 min
Figure 17. Oligonucleotide Dynamic Capacity Comparison:Aldehyde Reductase Primer (25 base) ± Trityl group
ProSwift WAX-1X (4.6 × 50 mm)300–925 mM NaCl in 18 min
0 7.5 15.0 22.5Minutes
We used a 25 mer mixed-base oligo with, and without, the protecting trityl- group to compare the dynamic loading capacity of the ProSwift Monolith to that of the DNAPac PA100, using comparably sized columns. Based on these comparisons the ProSwift WAX monolith has much greater dynamic capacity (Figure 17).
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.1 1.00 10.0 100 1000µg Injected
ProSwift WAX-1S
Figure 18. Dynamic Loading Capacity of Anion-Exchange ColumnsDNAPac PA100 (4 × 50mm) and ProSwift WAX (4.6 × 50mm)
DNAPac PA100~ 5% of ProSwift
Capacity v/v
Peak
Wid
th at
half
-heig
ht
Comparing the peak width (at half-height) of samples injected onto both columns, we observe a significantly better sample handling capacity on the ProSwift monolith. Based on these comparisons the ProSwift WAX monolith has 10–15 times the capacity of the DNAPac column, on a bed volume basis (Figure 18).
0 350
40
dA12
dA13
dA14
PdA1
2Pd
A13
PdA1
4Pd
A15
PdA1
6 PdA1
7Pd
A18
PdA1
9Pd
A20
PdA2
1Pd
A22
PdA2
3Pd
A24
PdA2
5Pd
A26
PdA2
7Pd
A28
PdA2
9Pd
A30
PdA4
0--
----
----
-- P
dA41
-- P
dA42
----
----
----
PdA
43--
PdA
44--
----
----
-- P
dA45
-- P
dA46
----
----
----
PdA
47--
PdA
48--
----
----
-- P
dA49
-- P
dA50
----
----
----
PdA
51--
PdA
52--
----
----
-- P
dA53
-- P
dA54
----
----
----
PdA
55--
PdA
56--
----
----
-- P
dA57
-- P
dA58
----
----
----
PdA
59--
PdA
60
1.25MNaCl: 32.0 %
54.0
75.0
Figure 19. Elution of PdA Oligonucleotides12- to 60-mers: ProSwift WAX
400–625 mM NaCl in 35 min, 20 mM Tris pH 8, 30 °C, Curve 4, 1 mL/min
The ProSwift WAX-1S column fully resolves very similar oligonucle-otides (here homopolymers of dA) up to 40 or more bases long, from their failure sequences.
TIDES2006Presentation �
In Figure 20, a repeat of the rA21 mer DNAPac study on the ProSwift WAX monolith, produced the same result. The undigested oligos elute in order of decreasing number of aberrant linkages. Also the elution positions of the digestion products are consistent with the number of consecutive aberrant linkages.
In Figure 21 a repeat of the rU21 mer DNAPac study on the ProSwift WAX monolith also produced the same result. The elution positions of the digestion products are consistent with the number of consecutive aberrant linkages.
In Figure 22 the new suite of mixed-base 21-mers with aberrant linkages was chromatographed on the ProSwift WAX-1S at pH 8. At this pH, samples Dio-5, Dio-6, Dio-9 and Dio-10 are completely resolved from all other samples, and are shown as red in the table. The pairs unre-solved at this pH are shown as black in the table.
0 240
180
PDAse-II Ctrl
rA(21)-PDAse-II
rA(21)L2-PDAse-II
rA(21)L4-PDAse-II
rA(21)L8-PDAse-II
rA(21)rA(21)L2rA(21)L4rA(21)L8
13.01 13.98
14.67
15.75
5.986.52
7.03
4.78
3.434.53 5.44
1.25M NaCl: 6.0 %
56.0
70.0
6.0
mA 26
0
Minutes
Figure 20. Elution of PDAse-II treated RNA on ProSwift WAX-1S:rA21mers, 75–700 mM NaCl/20 min, pH 8, 30 °C, 1 mL/min
PDAse-II Ctrl
rU(21)-PDAse-II
rU(21)L2-PDAse-II
rU(21)L4-PDAse-II
rU(21)L8-PDAse-II
rU(21)
16.43
5.676.33
6.97 7.57
4.234.98
5.86
3.45 4.43
1.25M NaCl: 56.0
70.0
Figure 21. Elution of PDAse-II Treated RNA on ProSwift WAX-1S:rU21 mers, 75–700 mM NaCl/20 min, pH 8, 30 °C, 1 mL/min
0 200
150
mA 26
0
Minutes
6.0 %
Dio-6:L2:AG10-12Dio-5:A10Dio-3:L2:AU1-3Dio-2:L1:A1-2Dio-1:L0Dio-8:L1:A5-6Dio-11:L2:C18-19,U20-21Dio-4:L2:A1-2,G3-4Dio-12:L2:U19-21Dio-10:L1:U20-21Dio-9:L1:C15-16
11.60
9.09
7.44
7.44
7.297.22
7.21
6.61
6.56
5.97
5.13
1.25M NaCl: 40.0 %
56.0
70.0
Figure 22. Effect of pH on RetentionOligonucleotide Linkage Variants: pH 8
ProSwift WAX-1S: 450–650mM NaCl/10 min, 30°C, 1 mL/min
D i o - 1 : 5 ´ A * U * G * A A * C U U C A * G * G G U C * A G C * U * U* G 3 ´
0 Minutes–10
240
mA 2
60
Dio-
2:Di
o-3:
Dio-
4:
Dio-
8:
Dio-
10:
Dio-
12:
Dio-
11:
Dio-
9:
Dio-
6:Di
o-5:
12.5
Sample: UnresolvedDio-1: 8, 11Dio-2: 3Dio-3: 2Dio-4: 12Dio-5:Dio-6
Sample: UnresolvedDio-8: 1, 11Dio-9Dio-10: Dio-11: 1, 8Dio-12: 4
� ChromatographicAssessmentofOligonucleotidePurity
North America
Sunnyvale, CA (408) 737-8522 Westmont, IL (630) 789-3660 Houston, TX (281) 847-5652 Atlanta, GA (770) 432-8100 Marlton, NJ (856) 596-0600 Canada (905) 844-9650
Europe
Austria (43) 1 616 51 25 Belgium (32) 3 353 4294 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 210 Italy (39) 06 66 51 5052 The Netherlands (31) 161 43 43 03 Switzerland (41) 62 205 99 66 United Kingdom (44) 1276 691722
Asia Pacific
Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 28475235 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580
LPN 1873-01 07/06©2006 Dionex Corporation
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www.dionex.com
In Figure 23 the same samples are chromatographed on the ProSwift WAX-1S at pH 9. All components not resolved at pH 8 are resolved at pH 9 so they are depicted in red in the table. Where the DNAPac PA200 required several pH values to resolve all of these components, the ProSwift monolith resolved them at only 2 pH values.
PROSwIfT wAX CONCLUSIONSProSwift WAX:
• Exhibits >10 fold more capacity per bed volume, than the DNAPac PA100 column.
• Can resolve oligonucleotides of 40 or more bases.• Retains mixed-base RNA in a pH-dependent manner (as with DNA).• Resolves mixed-base 21-mer linkage isomers, but may require dif-
ferent pH values to resolve all pairs.• Retains PDAse-II degradation products of each of the mixed-base
2´– 5´ linkage isomers to unique positions.• Is recommended for high resolution purification and analysis.• Will be available in larger sizes with a linear increase in sample
handling (up to ~ 5 mg on 10 × 100 mm format).
0 10
200
Minutes
10.64
8.14
7.51
7.01
6.92
6.62
6.50
6.25
6.11
5.98
5.18
mA 2
60
Dio-1:L0Dio-2:L1:A1-2Dio-9:L1:C15-16
Dio-12:L2:U19-21Dio-5:A10Dio-6:L2:AG10-12
Figure 23. Effect of pH on Retention ofOligonucleotide Linkage Variants: pH 9
ProSwift WAX-1S: 350–550mM NaCl in 10 min, 30 °C: 1 mL/min
0
Dio-4:L2:A1-2,G3-4Dio-3:L2:AU1-3Dio-8:L1:A5-6Dio-11:L2:C18-19,U20-21Dio-10:L1:U20-21
D i o - 1 : 5 ´ A * U * G* A A* C U U C A * G * G G U C * A G C * U * U * G 3 ´
Dio-
9:
Dio-
2:
Dio-
8:
Dio-
4:Di
o-3:
Dio-
6:Di
o-5:
Dio-
10:
Dio-
12:
Dio-
11:
Sample: UnresolvedDio-1: 8, 11Dio-2: 3Dio-3: 2Dio-4: 12Dio-5:Dio-6
Sample: UnresolvedDio-8: 1, 11Dio-9Dio-10: Dio-11: 1, 8Dio-12: 4
