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13360.41 (Δ 15 ppm)
13416.44 (+Fe)
13000 14000Mass
Analysis and Purification of RNAi and siRNA Sean M. McCarthy, Martin Gilar Waters Corporation, 34 Maple Street, Milford, MA 01757
OVERVIEW
• Oligonucleotides are a rapidly emerging class of biopharmaceutical compounds which present unique analytical challenges.
• Sequences are generated via high yielding step-wise synthesis, however they often require analysis and purification prior to use.
• There is a desire for MS new compatible mobile phases for single stranded and duplex oligonucleotide separations.
• New mobile phases should provide sufficient resolution of single stranded and duplex oligos with adequate MS compatibility.
• A systematic comparison of resolving power and MS compatibility of mobile phase modifiers for oligonucleotide separations is presented.
• UPLC/MS analysis of siRNA and its corresponding impurities and intact siRNA UPLC/MS analysis methods using new mobile phases are presented.
• Semi-preparative methods for purification of both single stranded and siRNA which are fully scalable are presented.
HOMOMERIC OLIGONUCLEOTIDE SEPARATIONS
0 11Minutes 0 11Minutes
100 mM
25 mM
0 11Minutes
100 mM
25 mM
0 11Minutes
100 mM
25 mM
0 11Minutes
100 mM
25 mM
0 11Minutes
100 mM
25 mM
TPAA
TEAA
DMBAA
TBAA HAA
TEA/HFIP
Figure 1: Separations were accomplished with a Waters ACQUITY UPLC® System using a Waters Oligonu-cleotide Separations Technology column (ACQUITY UPLC® OST C18, 1.7µm, 2.1x50 ) maintained at 60 °C. On column loading was 20 pmol/oligo of Waters MassPREP™ OST standard.
15 20
25 30
35
RNA DUPLEX ANALYSIS
• LC analysis of siRNA is difficult due to melting and incomplete resolution from mis-matched duplexes and other impurities.
• OST column technology coupled with HPLC and UPLC, using duplex compatible mobile phases, solves these problems by giving predictable retention and non-denaturing separation conditions.
• Use of IP RP HPLC and UPLC analysis of duplexes can be interfaced with MS to provide MS data unlike PAGE and AX-HPLC
Figure 3. Separation of mis-matched duplexes via UPLC with HAA mobile phase. ACQUITY UPLC® OST C18, 1.7µm, 2.1x50 mm column, 60 °C, UV 260 nm.
0 6
0 10
A
B
20° C
60° C
Purified Duplex
Crude Upper
Crude Lower
Purified Duplex
Crude Upper
Crude Lower
Minutes
Minutes
Figure 4: Raw and MaxEnt 1 deconvoluted spectra collected with single quadrupole detector from trun-cated siRNA separation shown in Figure 3. Data shown is for full length upper strand hybridized with N-1 lower strand (21u/20).
0 Minutes 25
ssRNAiImpurities
RNAi DuplexImpurities
85 nmol
15 nmol
1.5 nmol
0.15nmol
• Purification of siRNA is necessary to ensure specificity
• Purification accomplished by on-column annealing of siRNA • Inject first strand under initial gradient conditions immediately followed by injection of second complementary strand • After injection of second strand, gradient started to elute products
• Analysis of collected fraction confirms desired full length duplex composed of equimolar ratio of each complementary strand
Figure 8. (A) Semi-preparative purification of siRNA via on column annealing of complementary single stranded RNA (XBridge C18, 2.5µm, 4.6x50 mm, 20 °C). (B) and (C) verification of duplex purity at 20° C and 60° C respectively (ACQUITY UPLC® OST C18, 1.7µm, 2.1x50 mm).
A B
C
600 2000M/Z
-5
-4
MaxEnt 1
Lower6301.04
Upper6692.63
6000 7000M/Z
• Gradient adjusted so 15-mer eluted at 5 minutes and 35-mer at 10 min.
• Generally, the more hydrophobic IP agents (longer alkyl chain) required higher ACN content for oligo elution.
• Mobile phases prepared by from equimolar ratios of acetic acid and appropriate base, pH adjusted to ≈ 7.0
SINGLE QUADRUPOLE MS DETECTION
15 mM TEA
400 mM HFIP
100 mM TEAA
100 mM HAA
100 mM DMBAA
100 mM TPAA
100 mM TBAA
40
35
30
25
20
15
10
5
0
)/2w(wtt1P12
12
+−
+=
Peak C
ap
aci
ty
A
Figure 2: (A) Peak capacities for mobile phases at 100 mM for data shown in Figure 1. (B) Signal to Noise determined from three separate analysis of 25-mer oligo dT via single quadrupole MS.
16
8
0
TEA/HFIP
TEAA
DMBAATPAA
HAATBAA
S/N
(25-
mer
)
B
UV 260nm DETECTION
ssRNAi siRNA
Minutes0 8
N
N-1
Minutes0 5
Lower StrandPDII Digest
Upper Strand
siRNA PURIFICATION
21u
21
20
21u/2
1
21u/2
0
140 nmol
84 nmol
28 nmol
1.4 nmol
0 18Minutes
Purity ≈ 78%
0 6Minutes
Purified Oligonucleotide
Purity > 95%
*
N-1
1850 2350M/Z
1907.65 (-7)
2225.76 (-6)Raw MS data Only 2 charge states observed
CCC UGA GAU CCC AGC GCU G TT
M0 = 13360.2
TT GGG ACU CUA GGG UCG CGA C
Max
Ent 1
UPLC/MS ANALYSIS OF INTACT siRNA
TIC
UV 260 nm 4x
10x
Duplex
Duplex
Upper
Upper
Lower
Lower
0 8Minutes
Polarity ES-
Analyzer V Mode
Capillary 2.7 kV
Sample Cone 31.0 V
Extraction Cone 3.0 V
Source 150 °C
Desolvation 350 °C
Cone Flow 0.0 l/h
Desolvation Flow 800 l/h
Figure 6. Raw and MaxEnt1 deconvoluted data for intact siRNA analysis shown in Figure 5. Data acquired using Synapt HDMS mass spectrometer.
Figure 5. UPLC separation of siRNA. Mobile Phase A: 20 mM ammonium acetate, pH 6.5 B: Acetonitrile. ACQUITY UPLC® OST C18, 1.7µm, 2.1x50 mm, 20 °C, UV 260 nm.
Figure 7. (A) Semi-preparative purification of single stranded RNA (XBridge C18, 2.5µm, 4.6x50 mm, 60 °C ) fractions collected by heart-cutting of main peak. (B) Verification of purity at 60° C (ACQUITY UPLC® OST C18, 1.7µm, 2.1x50 mm, 60 °C).
RNA PURIFICATION
• Synthetic oligonucleotides are prepared by stepwise coupling
• Coupling efficiencies of 99.5% yield a 21 mer of approx. 90%
• Many applications require greater purity
Purified Oligonucleotide Purity >95%
N-1
A B