Download - Analysis and Purification of RNAi and siRNA...Analysis and Purification of RNAi and siRNA Sean M. McCarthy, Martin Gilar Waters Corporation, 34 Maple Street, Milford, MA 01757 OVERVIEW

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Page 1: Analysis and Purification of RNAi and siRNA...Analysis and Purification of RNAi and siRNA Sean M. McCarthy, Martin Gilar Waters Corporation, 34 Maple Street, Milford, MA 01757 OVERVIEW

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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).

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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

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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