Applications of Quantitative Mass Spectrometry Imaging in drug development David BONNEL, PhD ImaBiotech, France EBF 7th Open Meeting, Barcelona, 2014
Applications of Quantitative Mass
Spectrometry Imaging in drug development
David BONNEL, PhD ImaBiotech, France
EBF 7th Open Meeting, Barcelona, 2014
© ImaBiotech 2014
m/z Int.
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‘Nb of pixels’ = ‘nb of mass spectra’
Mass Spectrometry Imaging (MSI)?
© ImaBiotech 2014
m/z Int.
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Each m/z signal = One specific MS image
Mass Spectrometry Imaging (MSI)?
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WB Clozapine and its related metabolites distributions using MSI
Distribution of endogenous metabolites
Mass Spectrometry Imaging (MSI)?
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Distribution of endogenous metabolites
Mass Spectrometry Imaging (MSI)?
©2014 ImaBiotech
Fibrotic lung tissue
MSI and Histology
Glomeruli of kidney
Brain section
Amyloid plaques with Red Congo
Oil red O staining on skin tissue
MSI is a complementary technique to support histology
©2014 ImaBiotech
Bioanalytical Problematics
ANALYTICAL Variability
MALDI Matrix deposition
Mass Spectrometer deviation
BIOLOGICAL Variability
o Inter-/intra- tissue variation
o Ion suppression due to tissue/Ionization Yield
CALIBRATION CURVE
CC with criteria
LOD/LOQ Calculation
Absolute Quantification
Relative Quantification
©2014 ImaBiotech
QMSI workflow
Tissue
Section
Matrix
MSI
Process.
D Biological
D Analytical
D Analytical
D Analytical
CC Criteria
Dilution Range
« Pseudo » Internal Standard
Dosed Tissue
+
-
+
-
Quality Control
©2014 ImaBiotech
Applications ILC Small Molecule
Target molecule Diazepam
Model Dosed Rat (15 mg/kg, i.v.,
15 min)
Sample Brain
Therapeutic area Anxiolytic, anticonvulsant
Preparation Cryosection (10 µm)
Matrix DHB
ILC d5-Diazepam
(30 µM, m/z 290)
Ion images [M+H]+, m/z 285
Raster size 200 µm
Applications of QMSI
Instrumentation: MALDI-TOF Mass Spectrometer Autoflex Speed (Bruker Daltonics) Matrix Automated Deposition Device SunCollect (SunChrom)
©2014 ImaBiotech
Biological variability: Ion suppression on tissue
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Ventricle White Matter Hippocampus Thalamus Cortex
TEC
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ue
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Distribution of d5-Diazepam deposited on the brain & on the slide (200 µm)
Evaluate the specific ion suppression of each kind of tissues Tissue type specific Molecule specific
TEC = Int (Tissue) Int (Slide)
< 1
m/z 290.15
Image from QuantinetixTM
In triplicate
Reference Signal Cortex
Hippocampus
Ventricle
White Matter
Thalamus
©2014 ImaBiotech
Histological tissue effect: Intra-Normalization & “Real” Image
Normalization of molecular image using ILC
See the “real” distribution of the analyte1
Normalized image/d5-Diazepam Unnormalized image
m/z 285.06
Image from QuantinetixTM
Distribution of Diazepam within dosed brain section (200 µm)
©2014 ImaBiotech
Histological tissue effect: Intra-Normalization & “Real” Image
Normalization of molecular image using ILC
See the “real” distribution of the analyte1
Normalized image/Diazepam-d5 Unnormalized image
m/z 285.06
Image from QuantinetixTM
Distribution of Diazepam within dosed brain section (200 µm)
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Diazepam [M+H]+
m/z 285.06 S/N: 9
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Corronal Section Bregma -3.36 mm
m/z 283-287 m/z 283-287
Grey Matter
White Matter
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+ Diazepam Homogenate
(50 µg/g)
QC’s Sample preparation:
Frozen
Section
Mean 3 QC’s value
1 QC/image
1 QC/image
1 QC/image
Patent: WO 2012164221
Analytical variability: Inter-samples normalization
Replicate/Day 1 Replicate/Day 2 Replicate/Day 3
MSI
©2014 ImaBiotech
QC’s Control chart:
Patent: WO 2012164221
Analytical variability: Inter-samples normalization
QC’s Normalization Factor:
For inter-days/samples normalization, QCs…
Increase the reproducibility of MSI experiments
Provide better confidence into data interpretation.
QC = 1.02E+04
MS image n°
QCi (Int.) Coeff. = Qci / QC
1 1.09E+04 1.1
2 1.23E+04 1.2
3 7.52E+03 0.7
Normalization of MS images
©2014 ImaBiotech
Quantitation of Diazepam in Rat Brain using ILC
© ImaBiotech 2014
Small Molecule
Target molecules markers
Model IPF Rat Model (Bleomycin)
Sample Lung
Therapeutic area respiratory
Preparation Cryosection (10 µm)
Matrix DHB
ILC Matrix ions
Raster size 20 µm
Applications of QMSI
Instrumentation: MALDI-FTICR Mass Spectrometer Solarix (Bruker Daltonics) Matrix Automated Deposition Device SunCollect (SunChrom)
© ImaBiotech 2014
Readout Discovery: IPF Disease
Aim: identify some early readout markers of the IPF in a Bleomycin model
©2014 ImaBiotech
Readout Discovery: IPF Disease
0
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Fibrosis biomarkers intensity comparison between control (N=3) and IPF (N=6) tissues
Control
T7d
T22d
©2014 ImaBiotech
Conclusion
Analytical Variability = Quality Control (QC)
↑ Reproducibility/repeatability.
↓ Variation coeff.
Inter-samples comparison
↑ Confidence
Histological Variability = Internal standard (ILC)
Tissue Suppression Effect Normalization (Intra-)
See “real image”
ImaBiotech’s expertise: 200 QMSI projects
Success: 89% (limitation: sensitivity)
Average inter-sample variability: 15%-20% (based on the QCs)
Linearity: 2.5-3 Log
Best Limit of detection: 5ng/g
Average Limit of detection: 150ng/g
Quantinetix
Software
Multimaging Software
© ImaBiotech 2014
Dr. Jonathan Stauber Dr. David Bonnel Dr. Gregory Hamm Dr. Jean-Philippe Ebran Fabien Pamelard Alain Héron Raphaël Legouffe
Constance Buissart Guillaume Hochart Gael Picard-de-Muller Julien Stauber Emeline Falaux
Acknowledgement
Discovery Research Services, Edinburgh, United-Kingdom
Dr. Mary McELROY Dr. Stephen MADDEN
© ImaBiotech 2014
“A picture is worth a thousand words” Confucius (551–479 BC)
Thank you for your attention