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©2018 Waters Corporation 1COMPANY CONFIDENTIAL

Oligonucleotide Analysis SolutionsFrom Characterization to QC


Fit-for-purpose LC-MS workflows for oligo analysis:

Pages 3-19

Pages 20-41

Pages 42-57

Pages 58-63

Pages 65-76


Tools for Achieving Optimal

Oligonucleotide Separations


ACQUITY H-Class Bio UPLC System

Unrivaled UPLC resolution, sensitivity and throughput for optimum peak clarity and selectivity

Quaternary Solvent Manager (QSM) with Auto•Blend Plus™ for fully automated multi-

solvent blending

Bio-inert flow path with excellent corrosion resistance - uniquely suited for the high ionic strength aqueous conditions commonly used for biomolecular separations


Oligonucleotide Columns with BEH* Technology

ACQUITY UPLC columns for optimum speed,

performance and throughput

o BEH, C18, 130Å, 1.7µm Particles:

• 50 x 2.1 mm, 1.7 µm column

• 100 x 2.1 mm, 1.7 µm column

Xbridge™ HPLC/UHPLC columns for routine

analysis and lab scale purification

o BEH, C18, 130Å, 2.5µm Particles:

• 50 x 2.1 mm, 2.5 µm column

• 50 x 4.6 mm, 2.5 µm column

• 50 x 10 mm, 2.5 µm column

Patented *Bridged Ethyl Hybrid (BEH) Technology delivers N-1 oligo resolution

and superior column life at elevated pH values and temperatures


Longevity of BEH vs. Silica at High pH & Temperature

Accelerated High pH Stability Test of Competitive Columns

Silica Particles

BEH Particle Technology


MassPREP™ Oligonucleotide Standard

Note: Every batch of ACQUITY and XBridge Oligonucleotide column packing material is QC Tested with this standard to ensure performance and consistency

Pre-validated oligonucleotide reference material for calibration, troubleshooting and system suitability.

P/N 186004135


++

++ --

--

--

--+C18 sorbent

Ion Pairing Reverse Phase(IP-RP) Chromatography

Reversed Phase interaction withIon-Pairing reagent

-Hydrophobicity of the nucleobases + charge-charge interaction (oligo backbone)

Reversed-phase interaction withoutIon-Pairing reagent

- hydrophobicity of the nucleobases

0 minutes 10

1520

2535

30

0 minutes 10

1520

25

35

30

TEA+

TEA+ layer on the column surface


Ion Pairing Reagents for Oligonucleotide LC-MS Analysis

Gong, L. and J. S. O. McCullagh (2014). "Comparing ion-pairing reagents and

sample dissolution solvents for ion-pairing reversed-phase liquid

chromatography/ electrospray ionization mass spectrometry analysis of

oligonucleotides." Rapid Communications in Mass Spectrometry 28(4): 339-350.

TEA-HFIP was first ion pairing system for high efficiency LC-MS analysis of oligonucleotides

Apffel, A., et al. (1997). "Analysis of Oligonucleotides by HPLC−Electrospray Ionization

Mass Spectrometry." Analytical Chemistry 69(7): 1320-1325.

Waters scientists expanded on these capabilities over the following decade + of research

Apffel, A., et al. (1997). "New procedure for the use of high-performance liquid

chromatography–electrospray ionization mass spectrometry for the analysis of

nucleotides and oligonucleotides." Journal of Chromatography A 777(1): 3-21.

Gilar, M., et al. (2002). "Ion-pair reversed-phase high-performance liquid

chromatography analysis of oligonucleotides:: Retention prediction." Journal of

Chromatography A 958(1–2): 167-182.

Fountain, K. J., et al. (2003). "Analysis of native and chemically modified

oligonucleotides by tandem ion-pair reversed-phase high-performance liquid

chromatography/electrospray ionization mass spectrometry." Rapid Communications

in Mass Spectrometry 17(7): 646-653.

McCarthy, S. M., et al. (2009). "Reversed-phase ion-pair liquid chromatography

analysis and purification of small interfering RNA." Analytical Biochemistry 390(2):

181-188.


Range of Ion-Pairing Reagents Commonly Used Today

Ion Pairing Agent Buffering Acid Abbreviation

Triethylammonium Acetate TEAA

Triethylammonium Bicarbonate TEAB

Dimetylbutylammonium Acetate DMBAA

Tributylammonium Acetate TBAA

Tripropylammonium Acetate TPAA

Hexylammonium Acetate HAA

Triethylammonium Hexafluoroisopropanol TEA-HFIP

Selectivity and resolution impacted by choice

TEA-HFIP remains the most effective for single-stranded oligo analysis in

terms of LC-MS sensitivity and resolution, and is used throughout this

presentation


Triethylammonium HFIP Reagent

TriethylammoniumAcetate Reagent

0 30Minutes

13 14 15 1618

20

21 2226

29

25

28

30

27

24

23

19

171211

UV 2

60 n

m

10 32

24

11 12 13 14 15 16+17

18+19

20

21 2223

25

28

29

30

Minutes

UV 2

60 n

m

T

G

GG

C

G

A

TTT

C

C

TGTTAAGT

TEAA vs. TEA-HFIP for IP-RP Separation of an Oligo Mixture


Recent work: Alternative HFIP LC/MS Buffer systems

400 mM HFIP, pH 8.0

25 mM HFIP, pH 9.5

50 mM HFIP, pH 9.0

Triethylamine

Butylamine

Dibutylamine

Cost

$

$$$$$

$$$ 10x less

20x less

“standard”


Injection # 48 98 153 203 248 317 365 429Peak 4/5

Peak 3/5

Peak 2/5

Peak 1/5

BEH Columns Perform well under High pH

BEH, C18, 135Å, 1.7µm50 x 2.1 mm column

buffer exchange

Peak 4/5

Peak 3/5

Peak 2/5

Peak 1/5

Relative retention time to peak 5 (35 nt)15mM BA:50mM HFIP, pH 9.0

1 2 3 4 5

Stable chromatography over 400 injections


Peak Width (W50)

Inter assay Intra assay

Mean < 0.08 min 0.08 min

S.D. < 0.003 0.002

R.S.D (%) < 3.5 2.49

Ruggedness of BEH Columns

Average Peak width (W50) vs. Hours at pH 9.0

stable column performance in harsh conditions

Hour 29, inj # 53

Hour 124, inj # 243

Hour 193, inj # 421

15nt 20nt 25nt 30nt 35nt

N-1

N-1

N-1

Highly Reproducible and robust column performance over 200 hours at pH 9.0, 60 C using butylamine/ 50mM HFIP buffer


Troubleshooting Oligonucleotide LC-MS

- Quality of Mobile Phase Matters!

LC-MS grade solvents and

additives should be used

MS-grade TEA purchased from

Sigma (Fluka 65897-50ML-F)

MS-grade HFIP purchased from

Sigma (Fluka 42060-50ML)


Troubleshooting Oligonucleotide LC-MS

- Mobile Phases Need to be Fresh!

We recommend mobile phases be prepared daily to prevent MS signal loss

8.84E7

0.2556

5.87E7

0.2563

1.13E7

0.2609

1st injectionBase-line

+ 9 hours~30% drop in MS response

+ 2.5 days~87% drop in MS response

Remake BufferRecover MS response

1.43E8

0.213

TUV

MS

TIME


Troubleshooting - System Cleanliness Matters!

Contaminatedsystem

Clean system

Contaminatedsystem

Clean system

Systems previously used for aqueous based separations may require a multi-step cleaning

protocol. LC-HRMS/HDMS systems will require extended flushing with 50:50 H2O:MeOH, 0.1%FA

to reduce phosphate adduct from cleaning procedure


An effective protocol for reducing metal adducts

Reduction of metal adducts in oligonucleotide

mass spectra in ion-pair reversed-phase

chromatography/ mass spectrometry analysis

Robert E. Birdsall, Martin Gilar, et.al. Volume 30,

Issue 14 | 30 July 2016 (pages 1667–1679)

Describes an intermittent wash strategy that

prevents the accumulation of metal adducts over

time, which can cause MS signal degradation and

inconsistent performance.


Waters Oligo Applications Notebook

Download @ waters.com/oligos

Contains 24 application notes that demonstrate

the oligonucleotide separations performance and

productivity that can be achieved using Waters

column chemistries, reagent standards, UPLC

systems, and LC-MS workflows:

•UPLC-MS

•UPLC-HRMS

•UPLC-HDMS


UPLC-MS

Screening for Oligo ID and Purity

with the ACQUITY QDa Mass Detector

Our most deployable and scalable solution

Easy-to-use with a low maintenance requirement

Quickly confirm product ID & assess purity


Empowering analytical chemists and

chromatographers everywhere with

orthogonal mass detection

- see what lies under every peak

Innovative compact design focused on ease-

of-use, robustness and reliable performance

Seamlessly integrates into Empower CDS for

deployment in regulated & non-regulated labs

The ACQUITY QDa Detector


As Easy to Deploy as an Optical Detector

+ QDa

QDa Mass Detector

Empower® and MassLynx™ Control

Minimal operator training required

Qualification Service available

110/220V operation – no need for special outlet

Workhorse with low maintenance requirement


Automated Start Up and Calibration

Resolution and calibration verified automatically with each start-up ensures

mass data is accurate and reproducible. No tuning required!

ESI source optimized for UPLC/UHPLC

Graphic QDa monitor

enables easy viewing

and adjustment of

system parameters


Familiar User Interfaces for Ease-Of-Use and Deployment

Interface just like that of a PDA

Minimal training needed

Quick addendum to current SOP

Brings added MS functionality

Ability to deconvolute data

Export to ProMass for assignments


Disposable Components for Easy Maintenance

Sample Aperture:

As easy to replace as a detector lamp

Capillary:

Pre-assembled

capillary -

no cutting or

assembly

required


A Tool for Development and Production

Greater insight into every peak

Accelerated method and process development

More sensitive and selective attribute monitoring

Streamlined workflows for improved productivity

Compact, robust, easy-to-use and ready to deploy


TUV

15 nt20 nt 25 nt

30 nt

35 nt

QDa Proof-of-Principle with MassPREP Standard

nt Avg. MW [M-4H]-4 [M-5H]-5 [M-6H]-6 [M-7H]-7 [M-8H]-8 [M-9H]-9 [M-10H]-10 [M-11H]-11 [M-12H]-12 [M-13H]-13 [M-14H]-14 [M-15H]-15 [M-16H]-16 [M-17H]-17

15 4,500.9 1124.2 899.2 749.1 642.0 561.6 499.1 449.1 408.2 374.1 345.2 320.5 299.1 280.3 263.8

20 6,021.9 1504.5 1203.4 1002.6 859.3 751.7 668.1 601.2 546.4 500.8 462.2 429.1 400.5 375.4 353.2

25 7,542.9 1884.7 1507.6 1256.1 1076.5 941.9 837.1 753.3 684.7 627.6 579.2 537.8 501.9 470.4 442.7

30 9,063.8 2265.0 1811.8 1509.6 1293.8 1132.0 1006.1 905.4 823.0 754.3 696.2 646.4 603.2 565.5 532.2

35 10,584.8 2645.2 2116.0 1763.1 1511.1 1322.1 1175.1 1057.5 961.2 881.1 813.2 755.0 704.6 660.5 621.6

green = observable charge states

QDa50 pmolon-column

Multiple charge states

observed per oligonucleotide

Detection readily observed

with low pmol column loads

Quantification based on UV

Channel with MS providing

mass confirmation

P/N 186004135


Robust Reproducible Performance

15 nt 20 nt25 nt

30 nt

35 nt

646.5

696.2

754.4

823.1

905.41006.2 1131.8

n=3 [M-8H]-8 [M-9H]-9 [M-10H]-10 [M-11H]-11 [M-12H]-12 [M-13H]-13 [M-14H]-14 [M-15H]-15

Expected m/z 1132.0 1006.1 905.4 823.0 754.3 696.2 646.4 603.2

Average m/z 1132.1 1006.1 905.4 823.1 754.3 696.2 646.5 603.4

Delta +0.1 0.0 0.0 +0.1 0.0 0.0 +0.1 +0.2

%RSD 0.01 0.01 0.00 0.00 0.01 0.01 0.02 0.01

All data within QDa mass accuracy specification: +/- 0.2

TIC Trace:50 pmolon-column

Observed charge states for 30nt oligo: (-8 to -14)


MaxEnt1

Deconvolution

Manual Deconvolution with MaxEnt1

30 nt35 nt

Calculated Average Mass of 30nt = 9,063.8 Da

9,064.0 Da

+Na

Deconvoluted

Spectrum

9,086.0 Da

Raw ESI

Spectrum

646.5

696.2

754.4

823.1

905.4

1006.21131.8

MaxEnt1 deconvolution available within MassLynx provides for manual

deconvolution of nominal mass and high resolution data.


Enabling Automated Data Workflows with ProMass

“Automated” deconvolution and reporting package

nominal mass and high resolution mass spec (HRMS) data

Enables higher throughput workflows

+

Znova™

deconvolution for

nominal mass data

(Quadrupole)

Respect™

deconvolution for high

res data (QTof) –

available with

Promass HR


ProMass Workflow

MassLynx

Acquisition

• Acquisition of LC-

MS(/MS) data

•Automatically calls

ProMass Bridge

ProMass

“Bridge”

• Transfers data from

MassLynx to ProMass for

automated batch

processing / reporting

ProMass Processing

and Reporting

• Automated deconvolution

• Assess / confirm target

mass and purity

• Impurity peak

assignments

• Report Generation


ProMass: Deconvolution Parameters

Peak width set to span the width at the base of most peaks from the ESI mass spectra. A

typical Peak Width value is 2-3 m/z units.

Merge Width defines the m/z window were intensities are summed (merged) to compute a

centroided value. Typically 10-25% of the Peak Width (e.g., 0.2-0.5 for 2 m/z unit peaks)


ProMass: Batch Processing Parameters

Results Tab Reporting Tab


ProMass: Report Generation

Summary

Report

ESI Mass

Spectrum

Deconvoluted

Spectrum

Magnified

Deconvolution

Deconvolution

Peak Reports

Note:• Requires dongle to process (can not remote in)

• A top-level summary report shows results from multiple runs and provides hyperlinks that allows users to drill down into sample-specific results

• Target mass features allows one to automatically search for one or more target masses from the LC-MS data


ProMass: ZNova Mass Accuracy

n=3 nt 15 nt 20 nt 25 nt 30 nt 35

Expected Mass (Da)

4,500.9 6,021.9 7,542.9 9,063.8 10,584.8

Observed Mass (Da)

4,500.8 6,022.0 7,543.3 9,063.8 10,585.3

mass -0.1 0.1 0.4 0.0 0.5

SD 0.06 0.17 0.20 0.10 0.06

15 nt20 nt

25 nt

30 nt35 nt50 pmol

on-column 2 min window centered

around each peak used

for combined spectra

QDa mass data

deconvoluted with

Promass Znova™

algorithm

Mass errors within 1.0

Da of expected


QDa Enabled Mass Confirmation & Impurity Profiling

Upper Strand (50 pmol)

TUV, 260 nm QDa, 410-1250 m/z

Upper Strand, MW 6693.1 Da

5’-UCGUCAAGCGAUUACAAGGTT-3’

Lower Strand, MW 6607.0 Da

5’-TTCCUUGUAAUCGCUUGACGA-3’

Sample: Two distinct 21nt ssRNA oligos representing the complementary upper and

lower strands of a siRNA duplex (10 pmol/uL)

Time(min)

Flow (ml/min)

A B C D

Initial 0.200 82.0 18.0 0.0 0.0

4.00 0.200 80.0 20.0 0.0 0.0

4.01 0.200 50.0 50.0 0.0 0.0

6.00 0.200 50.0 50.0 0.0 0.0

6.01 0.200 82.0 18.0 0.0 0.0

4 Minute Gradient: 0.5%/min


Summary Report - Batch Results with Interactive Display

Target Mass: 6693.1 Da

(Upper Strand)

Data for 48 samples

processed in < 10 min

100% match with

expected results -

displayed in easy-to-use

color-coded format

Multiple target masses

can be searched

96-well plate format

Click for interactive sample report (following slide)

©2016 Waters Corporation 38

Interactive Sample Report

Total Ion Chromatogram

Blue Text = Hyperlinks to data views

Raw ESI Spectrum Deconvoluted Spectrum


Retention Time and Mass Accuracy

2

2.1

2.2

2.3

2.4

2.5

2.6

A1

A2

A3

A4

A5

A6

A8

B1

B2

B3

B4

B5

B7

B8

C1

C2

C3

C4

C6

C7

C8

D1

D2

D3

D5

D6

D7

D8

E1

E2

E4

E5

E6

E7

E8

F1

F3

F4

F5

F6

F7

F8Rete

nti

on

tim

e (

min

)

Retention Time (min) N=42Retention time (min)

Average 2.44

S.D. 0.01

%RSD 0.57

-1

-0.5

0

0.5

1

A1

A2

A3

A4

A5

A6

A8

B1

B2

B3

B4

B5

B7

B8

C1

C2

C3

C4

C6

C7

C8

D1

D2

D3

D5

D6

D7

D8

E1

E2

E4

E5

E6

E7

E8

F1

F3

F4

F5

F6

F7

F8

Mass e

rro

r (

Da)

Vial position

Mass Error (Da)N=42 Mass (Da)

Expected 6693.1

Average 6693.3

S.D. 0.3

Consistent retention

times across all 42

samples

All data within 1

Dalton of expected

mass


ProMass: Spectral Analysis for Purity

80

85

90

95

100

A1

A2

A3

A4

A5

A6

A8

B1

B2

B3

B4

B5

B7

B8

C1

C2

C3

C4

C6

C7

C8

D1

D2

D3

D5

D6

D7

D8

E1

E2

E4

E5

E6

E7

E8

F1

F3

F4

F5

F6

F7

F8Sp

ectr

al

ab

un

dan

ce

(%

)

Vial position

% Abundance in Spectrum

N=42 % Abundance

Average 94.11

S.D. 1.76

R.S.D. 1.87

A5

E6

N=40 % Abundance

Average 94.46

S.D. 0.79

R.S.D. 0.84

Non target masses other than predicted adducts are calculated as impurities


A) 5’-rUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGTT-3’, MW = 6,693.1 Da

B) 5’-C*T*C*T*C*G*C*A*C*C*C*A*T*C*T*C*T*C*T*C*C*T*T*C*T-3’, MW=7,776.3 Da

C) 5’-mU*mC*mG*mC*A*C*C*C*A*T*C*T*C*T*C*T*C*mC*mU*mU*mC-3’, MW=6,723.4 Da

Oligonucleotides – Detecting Modified Oligos

6 7 8 9 10 11 12

0.0

3.0x107

6.0x107

9.0x107

1.2x108

Inte

nsity

Upper

0.00

0.05

0.10

0.15

0.20

AU

Inte

nsity

A

U

n-1 n+1

n

TUVQDa

6,692.8 Da

6500 6550 6600 6650 6700 6750 6800 6850 6900

6 7 8 9 10 11 12

0.0

3.0x107

6.0x107

9.0x107

1.2x108

1.5x108

Inte

nsity

GEM91

0.00

0.05

0.10

0.15

0.20

AU

Inte

nsity

A

U

n-1 n+1

n

TUVQDa

7,776.0 Da

7600 7650 7700 7750 7800 7850 7900 7950 8000

6 7 8 9 10 11 12

0.0

5.0x107

1.0x108

1.5x108

2.0x108

Inte

nsity

GEM 92

0.00

0.07

0.15

0.23

0.30

AU

Inte

nsity

A

U

n-1n+1

n

TUVQDa

6500 6550 6600 6650 6700 6750 6800 6850 6900

6723.4 Da

A) siRNA unmodified B) Fully Thioated (PS) DNA C) 2’Ome Modified DNA

Mass information acquired with the QDa used to confirm product identity based on

average mass with excellent agreement between theoretical and experimental MW


UPLC-HRMS

Characterization and HRMS Monitoring

with the Xevo G2-XS Qtof

Robust High Resolution MS

Enhanced Sensitivity for Characterization Work

MS/MS for Sequence Confirmation


High Res Performance with MassPREP Standard

Instrumentation

LC: Waters ACQUITY® H-Class Bio

MS: Xevo G2-XS Qtof

Column Chemistry

ACQUITY UPLC OST BEH C18 Column 130Å, 1.7 µm, 2.1 mm X 50 mm (P/N: 186003949)

Waters MassPREP Oligonucleotide Standard

Reconstituted to 5 pmol/µL in pure H2O

1 µL of sample injected /LC-MS analysis

Method

15 min Ion Pairing RP gradient (TEA/HFIP)

Data collected by TUV and by ESI- MS


MassPREP Standard: TUV and TIC comparison

Load: 5 pmol on-column

TUV

TIC


MassPREP Standard - Raw ESI Spectra for 25nt Oligo

4-

3-5-6-7-8-

9-

10-

11-

12-

M3- charge state isotopic distribution

Resolution: > 30K

5 pmol on-column

+Na+

2508 2530m/z


Promass HR: Respect™ Deconvolution Mass Accuracy

Deconvolutedspectrum for 30nt

OST StandardExpected

MassObserved

MassMass Accuracy

(ppm)

15nt 4498.7348 4498.7300 -1.07

20nt 6018.9650 6018.9730 1.33

25nt 7539.1952 7539.1990 0.50

30nt 9059.4254 9059.4210 -0.49

35nt 10579.656 10579.6260 -2.79

Avg. 1.24

Mass accuracy: < 5 ppm

Promass HR includes the Respect™ deconvolution algorithm for processing high res MS data


MassPREP Standard - Determining Limit of Detection (LOD)

TUV

TIC

Load: 20 fmol on-column

Detection limited by UV signal, not MS sensitivity


Evaluating performance with ssRNA strands

Upper Strand, MW 6693.1 Da5’-UCGUCAAGCGAUUACAAGGTT-3’

Lower Strand, MW 6607.0 Da5’-TTCCUUGUAAUCGCUUGACGA-3’

1 µL injected for LC-MS analysis.

Sample: Two distinct 21nt ssRNA oligos representing the

complementary upper and lower strands of a siRNA duplex

(10 pmol/uL). 10 minute gradient for high resolution chromatogram

Oligo sourced from IDT


Upper Strand: 10 Min High-Resolution Chromatogram

N

N-1 N+1

N-1 = 5’-rUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 6386.9 Da; ProMass HR observed mass:6386.8 Da

N = 5’-rUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 6693.1 Da; ProMass HR observed mass:6693.0 Da

N+1 = 5’-rGrUrCrGrUrCrArArGrCrGrArUrUrArCrArArGrGrTT-3’Average mass = 7038.3 Da; ProMass HR observed mass:7038.7 Da

A ssRNA sequence 5’-UCGUCAAGCGAUUACAAGGTT-3’ with a double thymine overhang was separated from the base deletion (N-1) and base insertion (N+1) forms using a 10 min high resolution separation gradient from 13% B to 21% B.


Extracted Ion Chromatograms (XIC) of N-1, N and N+1

N+1

N (Target)

N-1XIC’s

time


Upper Strand: QTof ESI Raw Spectrum

4-

3-

5-6-

7-

8-

9-

M3- charge state isotopic distribution.

2199 2205m/z


Summary Report of Batch Results with Interactive Display

Promass HR Respect™

algorithmn used

Target Mass: 6689.9550 Da

(Upper Strand)

Data processed for all 48

samples in < 10 minutes

100% match with expected

results; displayed in easy-to-use

color-coded format


Interactive Sample-Specific Reporting

Total Ion Chromatogram Raw ESI Spectrum Deconvoluted Spectrum

Blue Text = Hyperlinks to Data Views


Oligo MS/MS Sequence Confirmation

Collision of oligonucleotides with

gas molecules at elevated energies

produces characteristic fragment

ions at each residue

McLuckey Fragmentation Scheme

c, y, & w ions predominate with QTof fragmentation


LC-MS/MS of m/z = 1650 (-4 Peak) Deconvoluted Spectrum[a] - , [b-H 2 O] - [b] -

[c] - , [d - H 2 O] - [d] - [a - B] -

225.052 243.062 305.018 323.029 113.024

530.093 548.103 610.059 628.070 419.050

875.140 893.151 955.107 973.117 724.091

1181.166 1199.176 1261.132 1279.142 1069.138

1486.207 1504.217 1566.173 1584.184 1375.164

1815.259 1833.270 1895.226 1913.236 1680.205

2144.312 2162.322 2224.278 2242.289 2009.257

2489.359 2507.370 2569.326 2587.336 2338.310

2794.400 2812.411 2874.367 2892.377 2683.357

3139.448 3157.458 3219.414 3237.425 2988.398

3468.500 3486.511 3548.467 3566.477 3333.446

3774.526 3792.536 3854.492 3872.503 3662.498

4080.551 4098.561 4160.517 4178.528 3968.524

4409.603 4427.614 4489.570 4507.580 4274.549

4714.645 4732.655 4794.611 4812.622 4603.601

5043.697 5061.708 5123.663 5141.674 4908.643

5372.750 5390.760 5452.716 5470.727 5237.695

5717.797 5735.808 5797.763 5815.774 5566.748

6062.844 6080.855 6142.811 6160.821 5911.795

6366.890 6384.901 6446.857 6464.867 6256.842

[w] -[x] - , [w - H 2 O] - [y] -

[z] - , [y-H 2 O] -

6462.888 6444.873 6382.922 6364.911

6157.847 6139.831 6077.880 6059.870

5812.799 5794.784 5732.833 5714.823

5506.774 5488.759 5426.808 5408.797

5201.733 5183.718 5121.767 5103.756

4872.680 4854.665 4792.714 4774.704

4543.628 4525.613 4463.662 4445.651

4198.581 4180.565 4118.614 4100.604

3893.539 3875.524 3813.573 3795.562

3548.492 3530.477 3468.526 3450.515

3219.439 3201.424 3139.473 3121.462

2913.414 2895.399 2833.448 2815.437

2607.389 2589.374 2527.423 2509.412

2278.336 2260.321 2198.370 2180.359

1973.295 1955.280 1893.329 1875.318

1644.243 1626.227 1564.276 1546.266

1315.190 1297.175 1235.224 1217.213

970.143 952.127 890.176 872.166

625.095 607.080 545.129 527.118

321.049 303.034 241.083 223.072

17.003 -1.012 -62.963 -80.974

Most C, Y, & W ions are matched

5 pmol on-column


LC-MS/MS Sequence Confirmation - Upper Strand

y2

c2

w2

y3

c3

w3

y4

c4

w5

y5

c5w4

y6

c7

w7

y7

w6

y8c9

y9

c8

y11 y12y10

c10

c16

w13

c15

c12 y14c14

Y17

y19c18

y20

-G

-G

M

y16

y18

c6

w8

w9w10

Y13

-G

w14

U C G U C A A G C G A U U A C A A G G T T5’- -3’

5 pmol on-column

*Deconvolution using MaxEnt-3


UPLC-HDMS

Characterization with Ion Mobility using the

SYNAPT G2-Si QTof

Ion Mobility brings an added dimension of separation that provides greater

spectral clarity for enhanced selectivity and sensitivity, especially for low level

impurities, and it enables 3D structure analysis


Techniques that can provide enhanced sensitivity and selectivity

Charge State / Drift Time Stripping

– Reduces spectral complexity; makes data more suitable for deconvolution

– Observe fragment ion series for each IMS resolved charge state

Wideband Enhancement

– Improve the resulting signal intensity (ca. 5-10 fold) of MS/MS spectra for low level

impurities

Time Aligned Parallel (TAP) Fragmentation

– Separation of primary fragment ions via ion mobility enables selection of specific primary

fragment ions for secondary fragmentation - Pseudo MS3

Ion Mobility Spectrometry (IMS)


Charge State / Drift Time Stripping

Analysis of PEG 4450 via HDMS

DriftScope™ data showing the gas-

phase separation power of the SYNAPT

HDMS System. Components with

different charge states are separated

via ion mobility

Waters Application Note: Characterizing Polyethylene

Glycol (PEG) by SYNAPT High Definition Mass

Spectrometry. Weibin Chen et.al.

Mass spectrum showing the ions with

+2 charge state. Improved mass

spectral clarity makes data more

suitable for charge state

deconvolution algorithms.


Wideband Enhancement


Time Aligned Parallel (TAP) Fragmentation


In Summary: LC-MS Workflows for Oligonucleotide Analysis

UPLC-MS ACQUITYQDa

HPLC-HRMSXevo G2-XS QTof

UPLC-HDMSSYNAPT G2 Si QTof

Mass Accuracy –Deconvoluted Data +/- 1.0 Dalton

< 5 PPM (~0.038 Da)

< 5 PPM (~0.038 Da)

Typical Column Load 50 pmol 5-10 pmol 5-10 pmol

Limit of Detection < 10 pmol 20 fmol 20 fmol

Mass Confirmation

Impurity Profile

Impurity ID

MS/MS Sequencing

(in source)

Cost $ $$$ $$$$


Appendix 1


BioAccord Intact Mass Analysis EnhancementNew Target Mass Screening / Mass Confirmation Workflow

NEW


Oligonucleotide Synthesis Houses

– Confirm identity and purity of therapeutic modalities

DNA based Diagnostic Test Kit Providers

– Confirm identity and purity of primers and probes

DNA Barcoding Companies

– Confirm identity of oligo-barcodes

Therapeutic Developers

– Incoming QC inspection on outsourced oligos

Who Needs Compliance-Ready Mass Confirmation?


Compliance-ready data acquisition, processing and reporting

Target mass screening via Intact Mass Analysis workflow

– Sequence entry not required, just target mass(es) of

oligonucleotides, proteins and related impurities

o Peptides handled via Accurate Mass Screening Workflow – no

deconvolution

– New negative ion data processing enables oligo data

analysis/mass confirmation

o Deconvolution available with BayeSpray or MaxEnt1

New Target Mass Screening / Mass Confirmation Workflow


Compliance-Ready Mass Confirmation of Oligonucleotides


Enter Target Mass(es) into Component Table

expected average masses

MassPrep OST Oligo Standard P/N 186004135

Select Deconvolution Algorithm: Select Molecule Type & Parameters:

New

Choices


Processing Results for MassPrep OST Oligos

Average mass accuracy

error: 4.7 ppm

Compliance-

ready mass

confirmation of

oligos with very

good mass

accuracy


Component Table for a 25mer Phosphothioated (PS) Oligo

Fully

Phosphothioated

25mer Oligo

New isotopic

model for

deconvolution;

enhances mass

accuracy when

working with

phosphothioated

species


Processing results for a 25-mer PPT oligo

Mass

accuracy

error: 6.7 ppm


Compliance-Ready Mass Confirmation of Proteins


Component Table for three mAb’s

Enter masses for each mAb (target mass) into the component table

Select molecule type:


1

3

2

TIC

TUV

Raw Spectra

1. Injection heat map 2. Sample list 3. Identified components

4. TIC and TUV traces5. Raw & Deconvoluted spectra

4

5

Deconvoluted Spectra

Review Pane:

- (Sample A2)

Theoretical (Mock) Spectra

Centroid


With UNIFI version 1.9.9, a new Target Mass Screening capability is now

available within the Intact Mass Analysis workflow of the BioAccord.

This capability enables Compliance-Ready mass confirmation of

Oligonucleotides, Proteins and Impurities.

All data acquisition, processing and reporting is done within UNIFI software,

which offers user level security, secure data storage, and a complete audit trail

capability so customers can ensure data integrity when providing QC reports or

submitting regulatory filings.

In Summary


To Learn More Visit:

WWW.Waters.com/oligos

or Contact your local Waters Account Representative

http://www.waters.com/oligos

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