oligonucleotide analysis solutions - waters corporation › webassets › cms › library ›...
TRANSCRIPT
©2018 Waters Corporation 1COMPANY CONFIDENTIAL
Oligonucleotide Analysis SolutionsFrom Characterization to QC
©2018 Waters Corporation 2COMPANY CONFIDENTIAL
Fit-for-purpose LC-MS workflows for oligo analysis:
Pages 3-19
Pages 20-41
Pages 42-57
Pages 58-63
Pages 65-76
©2018 Waters Corporation 3COMPANY CONFIDENTIAL
Tools for Achieving Optimal
Oligonucleotide Separations
©2018 Waters Corporation 4COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 5COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 6COMPANY CONFIDENTIAL
Longevity of BEH vs. Silica at High pH & Temperature
Accelerated High pH Stability Test of Competitive Columns
Silica Particles
BEH Particle Technology
©2018 Waters Corporation 7COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 8COMPANY CONFIDENTIAL
++
++ --
--
--
--+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
©2018 Waters Corporation 9COMPANY CONFIDENTIAL
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.
©2018 Waters Corporation 10COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 11COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 12COMPANY CONFIDENTIAL
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”
©2018 Waters Corporation 13COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 14COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 15COMPANY CONFIDENTIAL
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)
©2018 Waters Corporation 16COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 17COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 18COMPANY CONFIDENTIAL
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.
©2018 Waters Corporation 19COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 20COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 21COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 22COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 23COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 24COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 25COMPANY CONFIDENTIAL
Disposable Components for Easy Maintenance
Sample Aperture:
As easy to replace as a detector lamp
Capillary:
Pre-assembled
capillary -
no cutting or
assembly
required
©2018 Waters Corporation 26COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 27COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 28COMPANY CONFIDENTIAL
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)
©2018 Waters Corporation 29COMPANY CONFIDENTIAL
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.
©2018 Waters Corporation 30COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 31COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 32COMPANY CONFIDENTIAL
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)
©2018 Waters Corporation 33COMPANY CONFIDENTIAL
ProMass: Batch Processing Parameters
Results Tab Reporting Tab
©2018 Waters Corporation 34COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 35COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 36COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 37COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 39COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 40COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 41COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 42COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 43COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 44COMPANY CONFIDENTIAL
MassPREP Standard: TUV and TIC comparison
Load: 5 pmol on-column
TUV
TIC
©2018 Waters Corporation 46COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 47COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 48COMPANY CONFIDENTIAL
MassPREP Standard - Determining Limit of Detection (LOD)
TUV
TIC
Load: 20 fmol on-column
Detection limited by UV signal, not MS sensitivity
©2018 Waters Corporation 49COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 50COMPANY CONFIDENTIAL
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.
©2018 Waters Corporation 51COMPANY CONFIDENTIAL
Extracted Ion Chromatograms (XIC) of N-1, N and N+1
N+1
N (Target)
N-1XIC’s
time
©2018 Waters Corporation 52COMPANY CONFIDENTIAL
Upper Strand: QTof ESI Raw Spectrum
4-
3-
5-6-
7-
8-
9-
M3- charge state isotopic distribution.
2199 2205m/z
©2018 Waters Corporation 53COMPANY CONFIDENTIAL
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
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Interactive Sample-Specific Reporting
Total Ion Chromatogram Raw ESI Spectrum Deconvoluted Spectrum
Blue Text = Hyperlinks to Data Views
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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
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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
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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
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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
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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)
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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.
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Wideband Enhancement
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Time Aligned Parallel (TAP) Fragmentation
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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 $ $$$ $$$$
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Appendix 1
©2018 Waters Corporation 65COMPANY CONFIDENTIAL
BioAccord Intact Mass Analysis EnhancementNew Target Mass Screening / Mass Confirmation Workflow
NEW
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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?
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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
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Compliance-Ready Mass Confirmation of Oligonucleotides
©2018 Waters Corporation 69COMPANY CONFIDENTIAL
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
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Processing Results for MassPrep OST Oligos
Average mass accuracy
error: 4.7 ppm
Compliance-
ready mass
confirmation of
oligos with very
good mass
accuracy
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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
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Processing results for a 25-mer PPT oligo
Mass
accuracy
error: 6.7 ppm
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Compliance-Ready Mass Confirmation of Proteins
©2016 Waters Corporation 74
Component Table for three mAb’s
Enter masses for each mAb (target mass) into the component table
Select molecule type:
©2016 Waters Corporation 75
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
©2018 Waters Corporation 76COMPANY CONFIDENTIAL
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
©2018 Waters Corporation 77COMPANY CONFIDENTIAL
To Learn More Visit:
WWW.Waters.com/oligos
or Contact your local Waters Account Representative