LCMS Based Quantitation of Intact Proteins for ... · of Intact Proteins for Bioanalytical...
Transcript of LCMS Based Quantitation of Intact Proteins for ... · of Intact Proteins for Bioanalytical...
LC/MS Based Quantitation of Intact Proteins for Bioanalytical Applications
Alex Zhu, Ph.D.
Agilent Technologies
Wilmington, DE
ASMS,2017-06
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
1
Outline
• Introduction on intact protein quantitation
• Agilent Solutions for intact protein quantitation (standard flow
UHPLC/6545XT)
• Quantitation of intact mAb in neat solution
• Quantitation of intact mAb in rat plasma
• Quantitation of antibody drug conjugate in rat serum
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
2
Current Techniques for Protein Quantitation in Biological Matrices
• Ligand-binding assays (LBAs)
- High sensitivity; high throughput
- Require suitable capture and detection reagents; less specific, can be
affected by the presence of anti-drug antibodies in the samples
• MS based
- High specificity; wide dynamic range; faster method development;
quantify multiple proteins simultaneously; quantify PTMs, degradation
products, metabolites simultaneously
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
3
MS Based Protein Quantitation Strategies
• MS based
• Peptide approach
- Digestion is time consuming resulting in limited throughput
- Digestion efficiency and reproducibility requires extensive evaluation
- Selection of surrogate peptide is not simple
- Optimization of MRM method requires a lot of work
- Loss of essential information of the intact protein can lead to ambiguous
quantification
• Intact approach
- Higher throughput
- Specificity allows differentiation between closely related proteoforms
- Offering unique possibility to quantify therapeutic or clinically relevant
metabolites as well as postdose or pose-translational modified proteins
- More accurate quantitation
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
4
Challenges for Intact mAb Quantitation Using LC/MS
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
5
Challenges Causes Possible Solutions
Sensitivity “Diluted” peak intensity due to
formation of multiple charge
states
More sensitive instruments;
ways to reduce number of
charge states
Specificity Interference from
endogenous IgGs
Sample preparation
techniques; middle-down
approaches; Higher res instr.
(>600k) with fast enough
acquisition rate for quant
Chromatography Limited separation efficiency
and bad peak shape due to
heterogeneity
Better column; Alternative
separation mechanisms
(HILIC, etc.)
Data Analysis Single charge state?
Summation of several charge
states? Deconvoluted
Spectrum?
Need evaluation
Example: J&J (Jun, 2016)
J&J
Sample
Intact mAb in Plasma (Sigma
SiluLiteMab and SiluMab, SiluMab is
heavy labeled and used as IS)
Affinity
Purification Magnetic Beads
Flow rate Standard flow, 0.4 ml/min
Mass Spec TripleTOF API 5600
Quant
analysis Using height of deconvoluted spectra
LLOQ 60 ng on-column (1 µg/ml with 60 µl
inj)
Spectrum at
LLOQ level
Yes (large mass difference >100ppm
at all levels)
Linear range 1 order (due to limit of binding
capacity on beads)
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
6
Example: Novartis (Jan 2017)
Novartis’ Article
Sample Intact InfliximAb (Remicade) in rat
serum
Affinity
Purification
LB-MSIA (Ligand Binding-Mass
Spectrometric Immunoassay)
Flow rate 0.2 ml/min
Mass Spec QE
Quant
analysis Using sum of EICs
LLOQ 6 ng on-column
Spectrum
at LLOQ
level
Yes
Linear
range
2 orders (0.1-10 µg/ml with 60 µl
injection)
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
7
6545XT Features for Large Biomolecule Analysis
• Excellent protein spectral clarity from ultra-low TOF vacuum (10E-8)
• One-click optimization for large molecules with SWARM autotune
• Capable of analyzing very large molecules, with a variable mass range
up to 30k m/z
• Ease of maintenance with vent-free capillary removal
• Protein performance verification at install, and includes quick-start
protein method
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
8
Intact Protein Raw Spectral Quality
50+ 49+ 48+
Intact NIST mAb Analysis (0.5 µg injection)
Glycosylated Intact
Excellent raw spectral quality to
detect and identify minor isoforms
such as loss of amino acids, minor
glycoforms, and other PTMs.
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
9
Intact Protein Deconvoluted Mass Accuracy
6/14/2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
10
MaxEnt Deconvolution
- 0.64 ppm
1.44 ppm
- 0.06 ppm
0.78 ppm
- 3.72 ppm
G0F + G0F
G0F + G1F
G1F + G1F
G1F + G2F
G2F + G2F
3.54 ppm
(G0F+G0F)
- GlcNAc
(G0F+G1F)
- GlcNAc
(G1F+G1F)
- GlcNAc
(G1F+G2F)
- GlcNAc (G2F+G2F)
- GlcNAc
Intact NIST mAb Analysis (0.5 µg injection)
As seen in the raw spectra, the
increased spectral clarity leads
to improved deconvolved results,
both in accuracy and detection of
minor isoforms.
Maximum Entropy deconvolution
preserves low intensity signals
for investigation of heterogeneity.
How to Obtain Good Intact Protein (mAb) Spectra?
• Issues/complaints:
- Bad peak to hump
- Broad spectral peaks
- Loss of resolution of distinct
glyco-forms
- Bad mass accuracy
- Cleaning optics, changing
parts don’t help
- …
What’s Wrong??
June 14, 2017
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Procedures.
11
Key: Cleanliness of the LC!!! Check Your LC Carefully Before Blaming the MS
• Contaminants in the LC may not ionize, so won’t be seen
directly in the MS spectra, but could form complexes
(adducts) with the proteins, causing the issues!
- Contaminated bottles
- Buffer (water & organic)
- Tubing
- Column
- Valves
- Fittings/capillaries
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
12
One Example: Contaminants from Newly Opened HPLC Grade Bottled Water
Using Milli Q Water
Using HPLC Grade Bottled Water
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
13
Quantitation of Intact Herceptin In Neat Solution ASMS Poster: WP-637
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
14
Chromatography Optimization
Issue: Broad chromatographic peak, tailing, making it difficult for accurate quant at low levels.
From J&J Article
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
15
From Agilent app note (5991-6296EN)
Typical TIC for intact mAb, showing
board peak (>8s FWHM) and tailing.
Chromatography Optimization: Column Temperature (Herceptin)
RT
80 °C
FWHM = 11s with significant tailing
FWHM = 5.5s with some tailing
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
16
Chromatography Optimization (Herceptin)
Optimized Chromatography
FWHM = 5.5s with some tailing
FWHM = 2.1s with no tailing
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
17
Sensitivity and Linear Range of Glycosylated Intact mAb
6/14/2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
18
Trastuzumab
EIC Sum
Calibration Concentration (ng on-column)
0.0316 0.1 0.316 1 3.16 10 31.6 50
Accuracy (%, n=6) 102.5 94.1 95.1 95.6 101.7 110.4 105.4 95.4
Cal. Conc. %RSD (n=6) 7.40 3.83 5.48 4.37 6.56 2.20 0.68 2.68
Zoom in
Linear Range: > 3.2 orders
(31.6 pg50 ng or 200 amol350 fmol)
Standard Flow (0.4 ml/min)
Data from Trastuzumab Dilution Series
6/14/2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
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2x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
148053.51
148218.01
148379.79
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
3x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
148380.27
148221.24148062.96
148545.73
147911.38 148790.16148476.09
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 1489003x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
148221.02
148061.71 148380.15
148545.18
148486.09148280.42147917.18148150.08 148675.17
148855.77
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5 148220.09
148061.10 148382.80
148544.58
148320.56148469.44148149.73
147911.58148680.35
147992.93 148837.89147713.22
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 1489004x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8 148220.86
148381.80148059.52
148542.68
148316.78147912.51
148156.22148479.32
148703.37147852.26148830.20147705.77
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
148220.75
148382.11148059.51
148543.19
147913.28148317.32148156.92
148480.57148703.56147853.32 148004.83
147706.53 148832.85
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
5x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5148220.75
148381.86148059.73
148543.56
147913.20148318.84
148157.23 148480.89148705.04
147852.78 148003.31147707.42 148837.62
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 1489006x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1148220.64
148382.11148059.57
148543.22
147912.85148318.49
148159.05 148480.61148703.77147852.08 148002.42
147708.19 148835.66
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
0.0316 ng
0.1 ng
0.316 ng
1.0 ng
3.16 ng
10 ng
31.6 ng
50 ng
2x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
148053.51
148218.01
148379.79
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
3x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
148380.27
148221.24148062.96
148545.73
147911.38 148790.16148476.09
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 1489003x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
148221.02
148061.71 148380.15
148545.18
148486.09148280.42147917.18148150.08 148675.17
148855.77
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5 148220.09
148061.10 148382.80
148544.58
148320.56148469.44148149.73
147911.58148680.35
147992.93 148837.89147713.22
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 1489004x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8 148220.86
148381.80148059.52
148542.68
148316.78147912.51
148156.22148479.32
148703.37147852.26148830.20147705.77
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
148220.75
148382.11148059.51
148543.19
147913.28148317.32148156.92
148480.57148703.56147853.32 148004.83
147706.53 148832.85
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
5x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5148220.75
148381.86148059.73
148543.56
147913.20148318.84
148157.23 148480.89148705.04
147852.78 148003.31147707.42 148837.62
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 1489006x10
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
0.9
0.95
1148220.64
148382.11148059.57
148543.22
147912.85148318.49
148159.05 148480.61148703.77147852.08 148002.42
147708.19 148835.66
Counts vs. Deconvoluted Mass (amu)147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900
0.0316 ng
0.1 ng
0.316 ng
1.0 ng
3.16 ng
10 ng
31.6 ng
50 ng
Relative Abundances of Different Glyco-forms Across the Linear Dynamic Range (0.0316 - 50 ng)
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
20
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
Rel
ativ
e ab
un
dan
ces
of
maj
or
glyc
o-f
orm
s
50ng 31.6ng 10ng 3.16ng 1ng 0.316ng 0.1ng 0.0316ng
G0/G0F
G0F/G0F
G0F/G1F
G0F/G2F
G1F/G2F
G2F/G2F
Relative abundances of 6 major glyco-forms for
Herceptin at different levels (0.0316 – 50 ng)
Mass Accuracy Reproducibility (Accuracy and Precision) Across the Linear Dynamic Range (0.0316 - 50 ng)
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
21
-60
-40
-20
0
20
40
60
Mas
s Er
ror,
pp
m
G0F/G1F G0F/G0F G0F/G2F G1F/G2F G0/G0F G2F/G2F
0.0316 ng 0.1 ng 0.316 ng 1 ng 3.16 ng 10 ng 31.6 ng 50 ng
Excellent mass accuracy and reproducibility across the linear range
• For most abundant glyco-form G0F+G1F (148220.9758):
- Average mass error: -1.78 ppm
- Standard Deviation: 1.58 ppm
- Mass error range (-5.05 to 2.09 ppm)
Mass Accuracy Reproducibility (Accuracy and Precision) 100 Replicate Runs of 10 ng Injections
6/14/2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
22
10 ng on-column
Response Reproducibility 100 Replicate Runs of 10 ng Injections
6/14/2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
23
Norm
aliz
ed Inte
nsity
# of Runs
For 100 replicate runs, sum of EICs
of most abundant 12 spectral peaks:
- Standard deviation = 1.5% #40 Run
2.0s FWHM
Quantitation of Intact Herceptin in Rat Plasma
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
24
Acknowledgement: Kevin Bateman and Lisa Varicek (Merck) for
data acquisition and discussions
Experimental Flow
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
25
Rat plasma
(for serial
dilutions)
Trastuzumab
spiked Rat
plasma
SA-
W SA-
W
1290 Infinity II
6545XT AdvanceBio
LC/Q-TOF
MassHunter Quant
AssayMAP Bravo
Amount on Column
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
26
Starting
Concentration in
Plasma (ug/mL)
Initial Quantity in
30uL of plasma used
(ng)
AssayMAP Elution
Concentration
(ug/mL)
Amount on column
with 2uL injection
(ng)
0.05 1.5 0.06 0.12
0.1 3 0.12 0.24
0.5 15 0.6 1.2
1 30 1.2 2.4
5 150 6 12
10 300 12 24
25 750 30 60
100 3000 120 240
Calibration Curve EICs (Three at Each Level)
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
27
Calibration Curve
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
28
y = 33916x + 4748.5R² = 0.9999
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
2.5E+06
0 10 20 30 40 50 60 70
Zoom of bottom 4 points
0.0E+00
2.0E+04
4.0E+04
6.0E+04
8.0E+04
1.0E+05
1.2E+05
0 0.5 1 1.5 2 2.5 3
Agilent
Sample Intact Herceptin in rat plasma
Affinity
Purification AssayMAP
Flow rate Standard flow, 0.5 ml/min
Mass Spec 6545XT AdvanceBio Q-TOF
Quant
analysis Using sum of EICs
LLOQ 0.12 ng on-column
Linear
range 2.7 orders
Quantitation of Intact ADC (T-DM1) in Rat Serum ASMS Poster MP132
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
29
Calibration Curve for Deglycosylated T-DM1
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
30
Agilent
Sample Intact deglycosylated T-DM1 in rat
serum
Affinity
Purification AssayMAP
Flow rate Standard flow, 0.5 ml/min
Mass Spec 6545XT AdvanceBio Q-TOF
Quant
analysis
Using peak areas from
deconvoluted spectra
LLOQ 2 ng on-column for
deglycosylated T-DM1
Linear
range
2 orders (2-200 ng on-column,
assuming 100% recovery)
Example Spectra and DAR Calculation Deglycosylated T-DM1
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
31
2ng On-column 6.4 ng On-column
DAR Distribution at Different Levels
June 14, 2017
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Procedures.
32
Summary
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
33
Neat
Herceptin
Herceptin in Rat
Plasma
T-DM1 (ADC) in Rat
Serum
Sample Prep NA AssayMAP for affinity
purification
AssayMAP for affinity
purification and deglycosylation
Flow rate Standard flow, 0.5 ml/min Standard flow, 0.5 ml/min Standard flow, 0.5 ml/min
Mass Spec 6545XT 6545XT 6545XT
LLOQ 31.6 pg on-column
(Herceptin)
120 pg on-column
(Herceptin)
2 ng on-column
(Deglycosylated T-DM1)
Spectral
fidelity
Accurate glyco profile down to
LLOQ at 31 pg on-column
Accurate glyfo profile down to
LLOQ at 120 pg on-column
Accurate DAR calculation down
to LLOQ at 2 ng on-column
Linear range 3.2 orders (0.031 – 50 ng on-
column, Herceptin)
2.7 orders (120 pg – 60 ng on-
column, Herceptin)
2 orders (2 - 200 ng on-column,
deglycosylated T-DM1)
Summary
• 6545XT AdvanceBio QTOF coupled to 1290 infinity II
UHPLC provides you:
• Significant improvement on spectral quality
• Best sensitivity for intact mAb quantitation achieved using standard flow
• Best linear range
• Excellent reproducibility on both mass accuracy and response at
different levels including the LLOQ level
• Accurate measurement of glyco-form relative abundances across the
linear range including the LLOQ level
June 14, 2017
For Research Use Only. Not For Use in Diagnostic
Procedures.
34