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41
Quantitative Bioanalysis of Oligonucleotides Laixin Wang, Ph.D [email protected] May 24, 2016 Thermo BioSeparations Workshop Innovative Mass Spectrometric Solutions 1

Transcript of Quantitative Bioanalysis of Oligonucleotidestools.thermofisher.com/content/sfs/brochures/PP-O... ·...

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Quantitative Bioanalysis of Oligonucleotides

Laixin Wang, [email protected]

May 24, 2016Thermo BioSeparations Workshop

Innovative Mass Spectrometric Solutions

1

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Outline

• Overview

– Thermo instruments at NovaBioAssays

– Bioanalysis of oligonucleotide therapeutics

• DNApac PA200 columns for hybridization based LC-fluorescence assays

• DNApac RP columns for ion-pair LC-MS/MS and LC-HRAM analysis

– Full Scan, target-SIM and PRM/DIA analysis on Q-Exactive

• Q & A

2

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3

52 Dragon Court, Suite 3BWoburn, MA 01801, USAPhone: (781)933-3480

Where Are We

3

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What Do We Have (1/3)

Bruker MicroTOFQ II(nano and ESI source)

(x2)

Thermo Q Exactive MS (Plus)(x2)

Waters Acquity UPLCWaters nanoAcquity UPLC

(x7)

Thermo TSQ Vantage(nano and ESI source)

AB SCIEX 5000 LC/MS/MS(X2)

Accela 1250 and CTC PAL Thermo Easy nLC

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Thermo Vanquish Flex UHPLC System With Vanquish™ Fluorescence

Detector F (4 Excitation/Emission)

What Do We Have (2/3)

Waters Acquity UPLC with PDA

And Fluorescence Detectors

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What Do We Have (3/3)

6

Thermo KingFiser Flex 96Magnetic Particle Processor

Thermo Versette™ Automated Liquid Handler

TomTec Quadra 3 SPEAutomated Liquid Handler

(x2)

PPE, LLE, SPE Affinity/Immunoaffinity Purification

Mass Spectrometric Immunoassay (MSIA)

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Quantitative Bioanalysis (API5000 or Q-Exactive or FD/PDA)• Protein bioanalysis using LC-MS/MS or LC-HRAM

• Lipids/Polymers using LC-MS/MS or LC-HRAM

• Small molecule analysis using LC-MS or LC-HRAM

• Oligonucleotides bioanalysis using

LC-MS/MS or LC-HRAM

Hybridization UPLC fluorescence

UPLC-UV

Qualitative analysis (Q-Exactive and Q-tof)• Primary structural characterization using LC-HRAM and LC-MS/MS

• Tertiary structural characterization using H/D exchange LC-HRAM and LC-MS/MS

• Quant-Qual analysis using LC-Q Exactive

In Vitro Protein Binding and Stability/Metabolite ID• Rapid Equilibrium Dialysis(RED) and Ultracentrifugation

• Microsomal/Plasma/Whole blood stability

• Hepatocyte incubation

What Can We Do?

7

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Quantitative Bioanalysis of Oligonucleotides

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N

O B

PN O

O

O

OO

O B O

OHO

O B

LNA UNA

N

NH

O

B

O

PNA PMO

P

O

O OH PO OH

O

Chemical Structures of Oligonucleotides

9

O

O

O

O

O

O

R

PO

X-

B1

B2

R

X = O (Phosphodiester)S (Phosphorothioate)

R = -H (DNA)-OH (RNA)-OCH3 (2’-Methyl)-F (2’-Fluoro)-OCH2CH2OCH3 (2’-MOE)

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Quantitative Bioanalysis of Oligonucleotides/mRNA

10

• Hybridization-based assay (indirect analysis)

– Northern blot/in situ hybridization

– Microarray

– Deep sequencing (NGS)

– RT-qPCR

– Hybridization ELISA

• Direct ELISA, Sandwich ELISA, etc

• Branched DNA (bDNA) signal amplification technology (QuantiGene)

• Chromatographic-based assay (direction analysis)

– HPLC-UV/Fluorescence

– Hybridization-based LC-fluorescence

– LC-MS/MS and LC-HRAM

Analyte Oligo

Detection Probe

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Quantitative Bioanalysis of Oligonucleotides/mRNA

11

• Hybridization-based assay (indirect analysis)

– Northern blot/in situ hybridization

– Microarray

– Deep sequencing (NGS)

– RT-qPCR

– Hybridization ELISA

• Direct ELISA, Sandwich ELISA, etc

• Branched DNA (bDNA) signal amplification technology (QuantiGene)

• Chromatographic-based assay (direction analysis)

– HPLC-UV/Fluorescence

– Hybridization-based LC-fluorescence

– LC-MS/MS and LC-HRAM

Analyte Oligo

Detection Probe

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Ion-Pair (IP)-UPLC-UV Assays

• Simple sample preparation (single LLE or SPE)

• High reproducibility (15/20% acceptance criteria)

• Relatively selective (primarily by length/charge)

• Estimation of metabolite concentrations

• Reasonable run time (<10 minutes/sample)

• Low sensitivity (~100 ng/mL LLOQ)

Minutes

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

mA

u

0

100

200

300

mA

u

0

100

200

300

A

S1

411

Po

lyT

-30

Multi-Chrom 1 (1: 260 nm, 4 nm)10 44 S07169 20071690120 1004 AS1411 Day 6 URN-1 1

Name 26-mer

n-1

PolyT-30 Mobile Phase:20 mM tetrabutylammoniumsulfate in water/MeCNpH=9

Jian Chen, et. al., "Quantitative determination of AS1411 oligonucleotide in monkey plasma using UPLC-PDA," ISSX, 2008

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Strong Anion Exchange (SAX)-HPLC-UV

• Relatively selective(primarily by length/charge)

• High reproducibility(15/20% acceptance criteria)

• Simple sample preparation (single LLE or protease k digest)

• Estimation of metabolite concentrations

• Low sensitivity(~200 ng/mL LLOQ)

• Only works for charged oligos

• Long run time (20-35 min)

Sandberg, et al. Antisense & Nucleic Acid Drug Development 2000; 10:153-162.

Analyte: 35-mer modified ribozyme RNA

IS: 43-mer analog RNALC column: DNAPac® PA100MP: (50 mM Tris pH=8.0)/Ethanol80/20 (v/v) with 50-250 mM NaClO4

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Hybridization-based HPLC-Fluorescence Assays

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• High sensitivity (~ 1 ng/mL LLOQ )

• Good specificity (Hybridization and LC separation)

• Large dynamic range (1000x)

• Simple sample preparation (protein digestion or LLE)

• Good reproducibility (± 15% accuracy/precision)

• Tolerance to structure modifications Base, sugar and phosphate backbone modifications

3’

5

Sense

Anti-sense

Fluoro-probe

+3’

3

’5’ 5’

5’3’

1) 95ºC

2) 20ºC

HPLC

(SAX)

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Quantify siRNA in HuPl Using Fluorescence Labeled DNA Probe (1.00 – 500 ng/mL)

15Hutabarat R, et al. AAPS Annual Meeting 2011, poster presentation.

3’

5

Sense

Anti-sense

Fluo-probe

+3’

3

’5’ 5’

5’3’

1) 95ºC

2) 20ºC

Anti-

probe HPLC

(SAX)

Analyte: 21-mer siRNAProbe: 21-mer Atto dye labeled DNA oligoAnti-probe: Cholesterol conjugated DNA oligo

Sample Prep: Protease K digestionColumn: DNA Pac-PA-200, 4x250 mmMPA: 1 mM EDTA in 25 mM Tris-HCl (pH 8.0) with 10% MeCNMPB: 1 mM EDTA and with 800 mM NaCIO4 in 25 mM Tris-HCl

with 10% MeCN

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Typical Chromatogram of 22-mer siRNA in HuPl Samples (>24 Hours)

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22-mer siRNA

n-1

n-2

n-3

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Quantification of a Neutral 20-mer PMO Using a 15-mer Fluorescein-DNA

(40.0-4,000 ng/mL)

Arora V, et al. J of Pharmaceutical Sciences 2002; 91(4):1009-1018.

AVI-4126: 5’-ACGTTGAGGGGCATCGTCGC-3’ PMO (Phosphorodiamidate Morpholino Oligomer)

LC column: Dionex DNA Pac PA-100 (4 x 250 mm)Mobile Phase: A) 25 mM Tris pH=8.0;

B) 25 mM Tris pH=8.0 with 1.0 M NaCl

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Quantification of siRNA in HuPl Using Fluorescence-labeled PNA probe

(1.00-1000 ng/mL)

3’

5

Sense

Anti-sense

Atto-425

+3’

3

’5’ 5’

5’3’

1) 95ºC

2) 20ºCHPLC

(SAX)

Analyte: 21-mer siRNAProbe: 18-mer Atto dye labeled PNA oligo

Sample Prep: Protease K digestionColumn: DNA Pac-PA-200, 4x250 mm

Qingguo Tian, IBC’s 18th Annual TIDES May 9, 2016

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Method Performance of Quantitative Determination of

a siRNA in Rat Plasma using PNA Assay

R² = 0.9989

0.E+00

2.E+06

4.E+06

6.E+06

8.E+06

1.E+07

1.E+07

0 500 1000 1500

Pe

ak A

rea

Conc. (ng/mL)

LLOQ 1 ng/mLBlank

ISR Samples %Difference from original ISR samples

< ±10% 43

±10% ~ ±15% 2

Quality Control Samples

Incurred Sample Reanalysis

45 45 (100% pass)

QC (10 Runs) Accuracy (%Bias)

2.9%~11.2% -0.7%~2.5%

Precesion (CV%)

20%Difference from Orginal

Qingguo Tian, IBC’s 18th Annual TIDES May 9, 2016

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Pros and Cons of Hybridization-based HPLC-Fluorescence Assays

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• High sensitivity (~ 1 ng/mL LLOQ )

• Good specificity (Hybridization and LC separation)

• Large dynamic range (1000x)

• Simple sample preparation

• Internal standard is not necessary

• Good reproducibility (± 15% accuracy/precision)

• Tolerance to structure modifications

Base, sugar and phosphate backbone modifications

• Long run time (15-30 minutes/sample)

• One analyte/fluorophore at a time

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21

Thermo Vanquish Focused UHPLC System

With Vanquish™ FluorescenceDetector F (4 Excitation/Emission)

Chromeleon 7.2 Chromatography Data System

Fast and Mutiple Analyte Hybridizaionbased LC-Fluorescence Assays

DNApac PA200: 8 μm, (PEEK) 4.6 x 250 mm

DNApac PA200 RS: 4 μm, (Peek -lined 4.6 x 150 mmStainless Steel) 4.6 x 50 m

Data to be shared soon…

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LC-MS/MS & LC-HRAM Assays

22

• Highest specificity Molecular weight identification

Product ion characterization

LC Separation

• High throughput (~5 minutes/sample)

• Wide dynamic range (1000x)

• Good sensitivity (< 5 ng/mL LLOQ)

• No enzymes or special reagents needed

• Good reproducibility (±15/20% acceptance criteria)

• Need high performance mass spectrometer

• Potentially challenging assay development

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Challenges & Solutions of Developing LC-MS/MS Assays

23

• Non-specific adsorption (stickiness)– Add disruption reagent(s) in extraction buffer

– Avoid extensive drying down of samples

– Minimize use of polypropylene container

– Keep stock solution at high concentrations.

• Stability in matrix– Use K2EDTA as anticoagulant

– Add CHAPS or BSA to urine samples.

• Short column life time and carryover– Clean-up sample “appropriately”

– Re-generate the column after each injection (back flush the column).

• Charge distribution and metal ion adducts in mass spectrum– HFIP and TEA/DIPEA buffered water and MeOH/MeCN as mobile phase.

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Q1 MS Scan of a 18-mer PS-ODN on a Triple Quad

24

Calculated m/z

M 5731.6

M-6H 954.3

M-7H 817.8

M-8H 715.5

M-9H 636.8

-Q1: 20 MCA scans from Sample 1 (TuneSampleID) of MT20090528154857.wiff (Turbo Spray) Max. 6.6e6 cps.

620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960m/z, amu

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

Inte

ns

ity

, c

ps

708.9

715.6

818.0

636.0

954.5

625.2

736.9654.6

731.1

786.3673.2

663.0631.2641.0 746.2 771.2 920.9

627.3 717.0 936.9809.1705.4 857.3 915.1875.4820.7 836.8657.1 798.6643.1 758.2 899.0683.0661.2 772.8722.9 745.2 943.0921.7

Operator: Laixin Wang

Printing Date: Thursday, May 28, 2009

Results Name: N/A

M-9H

M-8H

M-7H

M-6H

Laixin Wang, et. al., AAPS Annual Meeting and Exposition, November 2009, Los Angeles, CA,

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Product Ion Scan of a 18-mer PS-ODN on a Triple Quad

25

-MS2 (715.40): 20 MCA scans from Sample 1 (TuneSampleID) of MT20090608140619.wiff (Turbo Spray) Max. 4.9e5 cps.

300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500m/z, amu

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e5

4.8e5

4.9e5

Inte

ns

ity

, c

ps

319.0

304.2

328.1

344.1

415.0346.0320.2400.1

321.9

329.2 424.0337.0305.1

384.0345.0 440.2357.1 494.8321.0 416.8 421.8362.1306.2 444.1399.0 408.3480.1382.1 445.1322.9 460.0332.3 485.1397.0348.2 437.8 464.3425.0369.6 410.1312.0 492.0

Operator: Laixin Wang

Printing Date: Monday, June 08, 2009

Results Name: N/A

SRM Transitions evaluated for TL0901

715.5 → 319.0

715.5 → 304.2

818.0 → 319.0

818.0 → 304.2

A

G

C

T

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LC Separation of a 18-mer PS-ODN and its Metabolites (Neat Mixture)

26

XIC of -MRM (3 pairs): 715.4/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 2.3e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min

0.0

1.0e4

2.0e4

In

te

ns

it

y,

c

ps

1.51

XIC of -MRM (3 pairs): 758.4/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 2.2e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min

0.0

1.0e4

2.0e4

In

te

ns

it

y,

c

ps

1.28

XIC of -MRM (3 pairs): 744.5/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 1.6e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min

0.0

5000.0

1.0e4

1.5e4

In

te

ns

it

y,

c

ps

1.41

XIC of -MRM (3 pairs): 715.4/319.0 amu from Sample 9 (25_09001_3 neat combo) of LCSET1.wiff (Turbo Spray) Max. 2.3e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5Time, min

0.0

1.0e4

2.0e4

In

te

ns

it

y,

c

ps

1.51

TL0901

TL0901 (n-4)

TL0901 (n-6)

n-4n-6

TL0901

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HRAM Improves Selectivity by Using Higher Resolution (Q Exactive)

27

1906 1907 1908 1909 1910 1911 1912 1913

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1909.18085

1909.85565

1908.52656

1910.17955

1907.78988

1907.430191910.51593

1910.83802

1911.18566

1909.51255

1909.18126

1908.84770

1909.84505

1908.52003 1910.17945

1910.51218

1908.191001910.83662

1907.793371911.18108

NL: 4.32E3

TL-Std5_1#377-390

RT: 1.69-1.75 AV:

14 T: FTMS - p ESI

SIM ms

[1907.30-1911.30]

NL: 7.32E3

TL-Std5_1#264-273

RT: 2.30-2.38 AV:

10 T: FTMS - p ESI

SIM ms

[1907.30-1911.30]

R=70,000

R=140,000

Isotope envelopes of the [M-3H]3- ion of an 18-mer PS-ODN on a high resolution Q-Exactive Orbitrap® (Thermo Finnigan, LLC)

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Different Charge Distribution of a18-mer PS-ODN on Different Platforms

28

-Q1: 20 MCA scans from Sample 1 (TuneSampleID) of MT20090528154857.wiff (Turbo Spray) Max. 6.6e6 cps.

620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960m/z, amu

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

In

te

ns

ity

, c

ps

708.9

715.6

818.0

636.0

954.5

625.2

736.9654.6

731.1

786.3673.2

663.0631.2641.0 746.2 771.2 920.9

627.3 717.0 936.9809.1705.4 857.3 915.1875.4820.7 836.8657.1 798.6643.1 758.2 899.0683.0661.2 772.8722.9 745.2 943.0921.7

Operator: Laixin Wang

Printing Date: Thursday, May 28, 2009

Results Name: N/A

M-9H

M-8H

M-7H

M-6H

Oligo_st_07 #495 RT: 2.22 AV: 1 NL: 5.28E6T: FTMS - p ESI Full ms [500.00-2500.00]

600 800 1000 1200 1400 1600 1800 2000 2200 2400

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

1909.1829

778.9326

1158.8998551.0519

968.91601200.8896

1431.6390

1348.88681948.2201

646.9078 817.6472 1052.89281864.16601532.8566 1989.77931686.7766

904.9360

2165.3530 2391.9988

M-3H

M-4H

Q-ExactiveAPI-5000

Weiwei Yuan, et. al. “Quantitative Determination of TL0901 Oligonucleotide in Human Plasma Using LC-HR/AM,“ ASMS, 2012

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-Q1: 1.407 to 1.525 min from Sample 15 (NBA1520_neat_50ugml_10ul_MS) of 20160203_381_257_HBV_TUNE.wiff (Turbo Spray) Max. 6.1e6 cps.

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m/z, Da

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.7e6

Inte

ns

ity

, c

ps

797.8

911.6

709.1

638.0

1063.9

541.4

746.3471.3 503.1547.2 609.8

781.0648.7 805.4757.4693.6525.2 563.1 574.3439.3 865.7654.4 1041.1

10-9-

8-7-

6-

API5000 Triple Quads

Different Charge Distribution of a20-mer PS-ODN on Different Platforms

Q Exactive Plus

NBA1520_FullMS_5ul #183-218 RT: 3.22-3.40 AV: 36 NL: 4.90E7T: FTMS - p ESI Full ms [600.0000-2500.0000]

600 800 1000 1200 1400 1600 1800 2000 2200 2400

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

2127.8546z=3

797.3193z=8

1595.6443z=4

1063.4284z=6

1276.3142z=5

2020.8549z=3

2276.9763z=3

3-

4-5-6-7-8-

9-

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Full Scan Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (1/3)

Extraction: SPE from 200 µL HuPl

LC Column:Thermo DNApac RP4 µm, 2x50 mm

Column Temp: 60 °CMobile Phase:

HFIP/TEA buffered water/MeOH Gradient

Run Time: 8 minutesLC System: Themo Vanquish UPLC

MS: Q-Exactive PlusIonization Mode: NegativeResolution: 70KScan Range 600-2500 m/z-

NBA1520: 5’--TAG TCA ATC TGC TTA TGT CA--3’ C196H249N68O102P19S19

NBA1519: 5’--TAG TCA ATC TGC TTA TGT C--3’ C186H237N63O98P18S18

NBA1514: 5’--TAG TCA ATC TGC TT--3’ C137H175N46O72P13S13

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31

Full Scan Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (2/3)

NBA1520: 5’--TAG TCA ATC TGC TTA TGT CA--3’ C196H249N68O102P19S19

NBA1519: 5’--TAG TCA ATC TGC TTA TGT C--3’ C186H237N63O98P18S18

NBA1514: 5’--TAG TCA ATC TGC TT--3’ C137H175N46O72P13S13

(A)

(B)(C)

(A): NBA1520

(B): NBA1519

(C): NBA1514

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Full-MS_STD210 #192-207 RT: 3.25-3.33 AV: 16 NL: 2.09E6T: FTMS - p ESI Full ms [600.0000-2500.0000]

600 800 1000 1200 1400 1600 1800 2000 2200

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1053.5355z=1

2127.8553z=3

962.9642z=2736.9414

z=1

926.9245z=1

2166.8953z=3

1595.6417z=4

1282.5730z=2

1116.9090z=1

1926.9323z=1

2083.1716z=3

1769.8316z=1

8-7-

4-

3-X

XX

X

Full Scan Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (3/3)

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NBA 1520 (Analyte)

LLOQ(0.5 ng/mL)

NBA 1514 (IS)

(200 ng/mL)

Extraction: SPE from 200 µL HuPlLC Column: Thermo DNApac RP

4 µm, 2x50 mmMobile Phase:

HFIP/TEA buffered water/MeOHLC System: Themo Vanquish Flux UPLC

MS: Q-Exactive PlusIonization Mode: NegativeResolution: 70Kt-SIM:

Analyte: 2123.8610-2131.8610IS: 1473.8065-1481.8065

Target-SIM Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (1/3)

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Target-SIM Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (2/3)

NBA 1520 (Analyte)

ULOQ(500 ng/mL)

NBA 1514 (IS)

(200 ng/mL)

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Target-SIM Analysis of Extracted 20-mer PS-ODN (HuPL) on Q-Exactive Plus (3/3)

Nominal C.

(ng/mL)

Average C.

(ng/mL) % Bias CV%

High QC 400. 395 -1.25% 4.51%

Medium QC 200. 193. -3.50% 5.29%

Low-Medium 15.0 14.2 -5.33% 7.10%

Low QC 1.50 1.41 -6.00% 4.48%

Carryover blank

(NBA 1520)

~21% of LLOQ

NBA 1514

(IS)

Curve range:

0.5-500 ng/mL

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Fragmentations of PS-ODN on Q-Exactivefor PRM/DIA Analysis Plus (1/2)

NBA1520_DIA2_160520143926 #421 RT: 3.26 AV: 1 NL: 3.16E7T: FTMS - p ESI Full ms2 [email protected] [301.0000-6000.0000]

1750 1800 1850 1900 1950 2000 2050 2100 2150 2200

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

-95.3694-50.3518

-45.0183

0.0000

-140.7185

-90.03541.3352

-190.7373-235.0587 -36.6804

-280.4053

2.3384

-102.7004

-153.0322

-330.7554 53.4163

-G

Precursor

2127.8611-A

-C

-AG

-GG

-AA

-AC

-GC

-AAG

-AAGG

-AGG

-AAAGG

-AAAGGG

5’--TAG TCA ATC TGC TTA TGT CA-3’

(NBA1520: A4C4G3T8)

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NBA1520_DIA2_160520143926 #425 RT: 3.27 AV: 1 NL: 6.11E5T: FTMS - p ESI Full ms2 [email protected] [302.0000-2470.0000]

310 320 330 340 350 360 370 380

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

346.0387

319.0165

337.0272328.0281

304.0168 325.0277

347.0417344.0230

329.0316348.0361305.0202 359.4888 379.4913372.5412

A

G

C

T

Fragmentations of PS-ODN on Q-Exactivefor PRM/DIA Analysis Plus (2/2)

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38

1) Aliquot 200 μl sample a) Add 50 μl IS to each wellb) Add 500 μl loading bufferc) Vertex briefly and spin down

2) Load the samples onto the SPE plate (Pre-equilibrated with 1 mL MeOH and 1 mL loading buffer)

3) Wash the plate with 1 ml loading buffer and 1 mL of washing buffer

4) Heat sample plate for 1 hr incubation at 70°C5) Elute the sample with 250-500 μl elution buffer

(contains 10% methanol)6) Inject 10 μl for LC-MS analysis

Extraction SPE Plate: 96-Well WAX SPE plate, 30 mg,

Sample Aliquot: 200 µL Human Plasma

Extraction Procedure:

SPE Extraction of the 20-mer PS-ODN From Human Plasma

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LC-MS/MS vs LC-HRAM

• Q1 mass spectra of oligos have different charge distributions on different instrument platforms

• LC-HRAM on Q-Exactive can achieve comparable sensitivity as LC-MS/MS on triple quads for some analytes.

• LC-HRAM can be performed without any prior knowledge of the compound, thus avoiding the need for compound-specific tuning (setting up the SRM transition). Therefore, it is very convenient for monitoring unknown metabolite information or estimate the known metabolite concentration(s) without standard reference material while quantifying the parent compounds.

39

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Size Maters for The Assay Sensitivities

40

Length of the oligonucleotide

10 20 30 40-mer

Sen

siti

vity

• LC-MS/MS (HRAM) assays are a good choice for small oligonucleotides;• Hybridization-based assays are advantageous for large

oligonucleotides.

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THANK YOU