Biopharmaceutical Attribute Monitoring with the Waters ACQUITY QDa Mass Detector

©2018 Waters Corporation 1COMPANY CONFIDENTIAL
Biopharmaceutical Attribute Monitoring
with the ACQUITY QDa Mass Detector
Bringing Greater Sensitivity, Selectivity and Productivity
to routine biopharmaceutical analyses

Before we begin…
The ACQUITY QDa is a Quadrople Mass Detector
three slides to make sure we’re all on the same page

MS DetectorUV Detectors
 Detects ions based on their mass to
charge ratio, or set of ratios (m/z values)
 the analyte must ionize or become
charged to be detected
How are UV and MS Detection Different?
 Measures absorbance of light at specific
wavelengths
 The analyte must have a UV
chromophore to be detected

Pre Filter
Quadrupole Filter
Produces nominal-mass data
The Quadrupole Mass Filter
 The quadrupole has four metal rods
 A combination of DC and Rf voltages separate,
or “filter” ions based on their m/z values
 At a given set of voltages only ions of a single
m/z value have a stable trajectory to the detector
 All others impact on the rods or are lost between them

Single Ion Recording
(SIR Mode)
Static
Full Scan
(MS mode)
Scanning
LOQ  500 pg (quantity injected)* LOQ  5 pg (quantity injected)*
Single Quad Modes of Acquisition
*compound dependent

 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

A Fit-for-Purpose Tool for Attribute Monitoring
Product & Process Monitoring / QC
Discovery & Characterization
“Mass Detection”
•Designed for max. accessibility
•Ease-of use
•Minimal training
•Compact size
•Affordable and Scalable
•Rugged, Robust Performance
“Mass Spectrometry”
•Most capable instruments
•Leading edge performance
•Expert level end users
•Large and not inexpensive
Synapt G2 Si
& VION HDMS
Xevo G2 XS
Non-Regulated Regulated - GXP
ACQUITY QDa
Future Offerings

QDa Biopharma Applications
Released N-Glycans
Polysorbate 80 / 20
Stability Monitoring
Oligonucleotides
ADC Free Drug Analysis
Synthetic PeptidesPeptide-based
Multi-Attribute-Monitoring
Confirm product identity (ID)
• Peptide Map / Profile
• CDR peptides
Post Translational
Modifications (PTM ) analysis
• Oxidation
• Deamidation, et.al.
• Glycopeptide Analysis
Sequence variants, …more

Peptide-based Multi Attribute Monitoring

Beginning with a Peptide Map…
Peptide-based multi-attribute-monitoring
(MAM) starts by running a peptide map:
• A tryptic “digest” of protein into a
unique set of peptides followed by a
reversed-phase (RP) separation
Shown is the “peptide map” for the mAb:
Trastuzumab using an Empower based
UPLC/TUV/QDa workflow with a CSH
C18 column*
The UV (top) and MS (bottom)
chromatograms captured from a single
sample injection within Empower.
*Reference: App Note 720005919EN, February 2017

Peptides well detected with TFA and FA Eluents
Heavy Chain Peptides from
a Trastuzmab digest
Trifluoroacetic
acid
Formic acid
= not observed
Legend:
>90 % Coverage

Greater Sensitivity & Lower Levels of Quantification
Dilution Conc (ug/uL) nanogram picomole
2 0.125 1025.00 6961.19
3 0.0625 512.50 3480.59
4 0.03125 256.25 1740.30
5 0.015625 128.13 870.15
6 0.0078125 64.06 435.07
7 0.0039063 32.03 217.54
8 0.0019531 16.02 108.77
9 0.0009766 8.01 54.38
10 0.0004883 4.00 27.19
11 0.0002441 2.00 13.60
12 0.0001221 1.00 6.80
13 0.0000610 0.50 3.40
14 0.0000305 0.25 1.70
15 0.0000153 0.13 0.85
16 0.0000076 0.06 0.42
17 0.0000038 0.03 0.21
18 0.0000019 0.02 0.11
19 0.0000010 0.01 0.05
20 0.0000005 0.004 0.03
21 0.0000002 0.002 0.01
Trastuzumab T21 Peptide Dilution Series*:
linear
QDa Detector Linearity (SIR Mode)
linear
TUV Detector Linearity
Dilution Conc. (ug/uL) nanogram picomole
2 0.125 1025.00 6961.19
3 0.0625 512.50 3480.59
4 0.03125 256.25 1740.30
5 0.015625 128.13 870.15
6 0.007813 64.06 435.07
7 0.003906 32.03 217.54
8 0.001953 16.02 108.77
9 0.000977 8.01 54.38
10 0.000488 4.00 27.19
11 0.000244 2.00 13.60
12 0.000122 1.00 6.80
QDa Detector Linearity (Full Scan)
Dilution Conc. (ug/uL) nanogram picomole
2 0.125 1025.00 6961.19
3 0.0625 512.50 3480.59
4 0.03125 256.25 1740.30
5 0.015625 128.13 870.15
6 0.007813 64.06 435.07
7 0.003906 32.03 217.54
8 0.001953 16.02 108.77
9 0.000977 8.01 54.38
10 0.000488 4.00 27.19
11 0.000244 2.00 13.60
12 0.000122 1.00 6.80
linear
The QDa adds sensitivity and extends the linear dynamic
range of detection, enabling lower levels of quantification,
especially in SIR Mode.
*(UPLC/TUV/QDa w/ CSH & 0.1% FA)

Peptide-based MAM: CDR Peptides for Antibody ID
The unique binding affinity of every antibody stems from its Complementary Domain
Region (CDR) - circled above. The CDR peptides from this region are unique to each
antibody, hence CDR peptide profiles are often used to confirm antibody ID.
Image source: School of Biomedical Sciences Wiki

Peptide Based MAM: Oxidation
Blind case study: digested mAb
sample and SIR m/z values provided:
Relative Quantification Results:
Peptide H21and H21-Ox
SIR m/z values
H21: M+2 [418.4]
H21-Ox: M+2 [426.4]
H21
H21-Ox
MS Inst. H21 H21-Ox
QDa 96.8% 3.2%
QTof 97.5% 2.5%

H37
0-65 min 65-140 min
H37-D
Peptide Based MAM: Deamidation
Peptide H37 and H37-D
MS Inst. H37 H37-D *
QDa 89.1% 10.9%
QTof 89.2% 10.8%
Blind case study: digested mAb
sample and SIR m/z values provided:
SIR m/z values
H37: M+2 [849.2]
H37-D: M+2 [849.6]
One SIR channel used with two time windows
*Combination of H37-D & H37-isoD

Peptide Avg. Mass
SIR
[M+3]
H25 1189.2 397.4
G0F 2633.7 878.9
G1F 2795.8 932.9
G2F 2957.8 986.9
G0 2487.7 830.2
G1 2649.7 884.2
Man5 2405.6 802.9
G0F
G1F
G2F
G0 G1Man5 H25
Zoom-in
QDa: SIR Mode
Peptide Based MAM: Glycopeptide Analysis
Blind study: digested mAb sample
and SIR m/z values provided:
MS G0F G1F G2F
Qda 51.1% 43.8% 6.1%
Qtof 50.2% 43.7% 6.2%

Monitoring Multiple Attributes in a Single Assay
Attribute Monitoring:
 CDR Peptides for ID
 % Oxidation
 % Deamidation
 Glycopeptide profile
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
SIR function: 1 2 3 4 5 6 7 8 9
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
-2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0
2
4
SimulatedY1
x0 = 0
dx = 0.15075376884422
Gauss
retention timem/z
888.4
444.7
spectrum
Supports compliance-ready monitoring
of multiple attributes in a single assay
Time-based SIR functionality used to
maximize sensitivity

Synthetic Peptides
Assess purity with greater confidence, detect co-eluting impurities in method development - use
results to optimize gradient, monitor production for early detection of synthesis failures, utilize
mass directed purification to increase purity and productivity, enhance QC with mass confirmation.
Original Gradient Optimized Gradient
Reference: App Note 720005967EN, April 2017

Released N-Glycan Analysis
 Routine Mass Detection of
RapiFluor-MS labeled N-
Glycans over a large
dynamic range.
 Glycan structure
confirmation via mass, with
improved quantification, and
faster/higher throughput
glycan monitoring methods.
With RapiFluor-MS:

Released N-Glycans: Wide Range of Glycoforms Detected
Minutes
5 10 15 20 25 30 35 40
Minutes
5 10 15 20 25 30 35 40
EU
0
20
40
60
FLR
Intensity
1x106
2x106
3x106
4x106
5x106
6x106
QDa
Equivalent FLR and MS responses from a single run. Confirm or reject glycan
assignments via mass. Achieve faster method development & screening.
IgG
Simple bi-antennary
structures
RNase B
High mannose
structures
Fetuin
Large, multi-antennary,
highly sialylated
structures
Overlay of three samples

TUV
15 nt
20 nt 25 nt
30 nt
35 nt
Oligonucleotides
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
QDa
50 pmol
on-column
 Multiple charge states
observed per oligonucleotide
 Detection readily observed
with low pmol column loads
 Run in negative ion mode;
quantification based on UV
absorbance - MS used for
mass confirmation
MassPrep™
Oligonucleotide
Reference Standard
P/N 186004135

Oligonucleotides - Robust, Reproducible Performance
15 nt 20 nt
25 nt
30 nt
35 nt
646.5
696.2
754.4
823.1
905.4
1006.2 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 pmol
on-column
Observed charge states
for 30nt oligo: (-8 to -14)

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
3.0x10
7
6.0x10
7
9.0x10
7
1.2x10
8
Intensity
Upper
0.00
0.05
0.10
0.15
0.20
AU
Intensity
A
U
n-1 n+1
n
TUV
QDa
6,692.8 Da
6500 6550 6600 6650 6700 6750 6800 6850 6900
6 7 8 9 10 11 12
0.0
3.0x10
7
6.0x10
7
9.0x10
7
1.2x10
8
1.5x10
8
Intensity
GEM91
0.00
0.05
0.10
0.15
0.20
AU
Intensity
A
U
n-1 n+1
n
TUV
QDa
7,776.0 Da
7600 7650 7700 7750 7800 7850 7900 7950 8000
6 7 8 9 10 11 12
0.0
5.0x10
7
1.0x10
8
1.5x10
8
2.0x10
8
Intensity
GEM 92
0.00
0.07
0.15
0.23
0.30
AU
Intensity
A
U
n-1
n+1
n
TUV
QDa
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

Antibody Drug Conjugate (ADC) Free Drug Analysis
Benefits
 No sample preparation required
 Sensitive MS detection
 SIR specificity
 No co-elution or matrix effects
ACD
Waters patented* 2D LC workflow:
1st Dimension:
Trap and elute from an
OASIS Max SPE column
At column dilution:
Reduce organic solvent
concentration
2nd Dimension:
Retain on Cortecs C18
HR Column
MS Detection w/QDa
* At Column Dilution Patent

ADC Free Drug Analysis: MS for Greater Sensitivity
Addition of the QDa increases sensitivity 150-fold compared to UV based techniques
(0.3 ng/mL versus 50 ng/mL respectively), allowing for a much lower level of quantification
Current FDA guideline:
Max allowable free drug = 0.1%
Current FDA guideline (0.1%)
Published UV limit
150 fold more sensitive
QDaTUV
TUV
LinearRangeQDa
LinearRange

Polysorbate 80 (PS-80) Stability Monitoring
Polysorbate 80 (PS-80), and other
non-ionic surfactants are used in
final formulations to prevent:
 Surface adsorption
 Air-water interface turnover
 Aggregation
 Denaturing
PS-80 degradation can negatively
impact drug safety & efficacy
Raw material often tested for
consistency and monitored for
degradation during stability testing
UPLC-RP/TUV/QDa Workflow
UV-active
Oleic acid
Non UV-active
Degradation by-product:
Free fatty acid (Oleic Acid) –
(unique to each surfactant);

Oleic acid (OA) detected in infliximab sample at a concentration of <1% relative to the internal
standard (IS) following base hydrolysis. Not detected in trastuzumab sample (as expected).
Results confirmed by mass detection using the ACQUITY QDa (inset).
PS-80 Stability Monitoring – Proof of Principle
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
0.00
0.01
0.02
0.03
0.04
0.05
A.U.
retention time (min)
B
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
0.00
0.01
0.02
0.03
0.04
0.05
A.U.
B
IS
OA OA
Infliximab (PS-80; Oleic Acid) Trastuzumab (PS-20; Lauric Acid)
TUV
IS
Retention time (min)
A.U.
Retention time (min)
A.U.
99.08%
0.92%
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
0.0
2.0x10
6
4.0x10
6
6.0x10
6
8.0x10
6
1.0x10
7
1.2x10
7
Intensity
SIR
MS (SIR)
IS
OA
QDa
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
0.0
2.0x10
6
4.0x10
6
6.0x10
6
8.0x10
6
1.0x10
7
1.2x10
7
ntensity
SIR
MS (SIR)
IS
OA
QDa
TUV

Growing Industry Adoption and Validation

Being Implemented Today in GMP Regulated Labs

To Learn More Please Visit:
www.Waters.com/BiopharmQDa
• Presentations
• Application Notes
• Posters
• Publications
• Additional Product Info, etc.

Biopharmaceutical Attribute Monitoring with the Waters ACQUITY QDa Mass Detector

Biopharmaceutical Attribute Monitoring with the Waters ACQUITY QDa Mass Detector

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