Analysis and Purification of RNAi and siRNA ... Analysis and Purification of RNAi and siRNA Sean M.

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Transcript of Analysis and Purification of RNAi and siRNA ... Analysis and Purification of RNAi and siRNA Sean M.

  • TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2008 Waters Corporation

    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

    ssRNAi Impurities

    RNAi Duplex Impurities

    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

    Lower 6301.04

    Upper 6692.63

    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

    HF IP

    100 mM

    TEA A

    100 mM

    HA A

    100 mM

    DM BAA

    100 mM

    TPA A

    100 mM

    TB AA

    40

    35

    30

    25

    20

    15

    10

    5

    0

    )/2w(w tt1P 12

    12

    + −

    +=

    P e a k C

    a p

    a ci

    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

    TE A/

    HF IP

    TE AA

    DM BA

    A TP

    AA HA A

    TB AA

    S/ N

    (2 5-

    m er

    )

    B

    UV 260nm DETECTION

    ssRNAi siRNA

    Minutes0 8

    N

    N-1

    Minutes0 5

    Lower Strand PDII Digest

    Upper Strand

    siRNA PURIFICATION

    2 1 u

    2 1

    2 0

    2 1 u /2

    1

    2 1 u /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

    M ax

    En t 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