free mmae toxin quantitation by triple quadrupole in...
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Free MMAE toxin quantitation by triple quadrupole in Antibody Drug Conjugate analysis
Proteomic Platform Innovation Technologic Timone PIT2 Shimadzu
Sega NDIAYE EBF Barcelona 2013
Overview
• Introduction
• Antibody Drug Conjugate • General • ADC Brentuximab-Vedotin • Monomethyl Auristatin E
• Equipment
• Methods
• Results
• Conclusion
ntibody
onjugate
rug
ADCETRIS
Brentuximab-Vedotin
cAC10 Chimeric IgG1
maleimidecaproyl Dipeptide Valine
Citrulline
p-aminobenzylcarbamate Monomethyl auristatin E
Monomethyl auristatin E
• The auristatin E synthetic analog of the natural product dolastatin 10
• Dolastatin 10 was originally isolated from the Indian Ocean sea hare, Dolabella auricularia
• High toxicity
• Blocks tubulin polymerization
MMAE Chemical structure Dolastatin 10 Chemical structure
Wedge sea hare
LC-MS/MS system
• SHIMADZU 8040 LC-MS/MS • Liquid Chromatography UHPLC
Nexera SHIMADZU
• Electrospray interface
• Triple quadrupole
• Speed : up to 555 MRM/sec
• Sensitivity : ≈ 1 fmol (Réserpine M=608.7 S/N 10000)
Methods
• MMAE protocol extraction
20µl plasma 5µl internal standard
MMAF spiking
Protein
Precipitation (50µL MeOH)
Centrifugation
Recovery supernatant
(40 µL) (in 80µL H2O/MeOH)
SPE (MMAE High toxicity)
LC-MS/MS Analysis
Triple quadrupole
Methods
• Chromatography • Kinetex XB-C18 Phenomenex UHPLC Column • Eluent : H2O = A et MeOH = B Flow rate : 0.7 mL/min T : 40°C • Total duration of chromatographic run : 8 min • Assay with internal standard: MMAF
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0 1 4 5,5 7 7,1 8
% o
rgan
ic p
hase
Minutes
Gradient
% Phase B
Methods
• Mass spectrometry triple quadrupole • Triple quadrupole using SRM (Single Reaction Monitoring)
Q1 Q2 Q3
SIM Fragmentation SIM
Single reaction monitoring
• Increase the signal to noise ratio
Detection
Methods • Mass spectrometry • 2 SRM transition by compound : MRM Mode
• MMAE : 718.50 → 686.50 ; 718.50 → 152.10 • MMAF : 732.50 → 700.30 ; 732.50 → 170.30 • Fragments verified by Product Ion Scan and literature search
Product Ion Scan MMAE Product Ion Scan MMAF MMAE
Transition Dwell time (ms) Q1 PreBias (V) Collision Energy (V) Q3 PreBias (V)
718,5->686,3 100 -28 -31 -34
718,5->152,0 100 -28 -36 -29
MMAF
Transition Dwell time (ms) Q1 PreBias (V) Collision Energy (V) Q3 PreBias (V)
732,5->700,3 100 -28 -29 -26
732,5->170,1 100 -28 -46 -30
Optimization
MMAE signal response optimization MMAF signal response optimization
Mobile phase optimization
Optimization
• Interface • Electrospray source parameters optimization (3 factors, 2 levels)
• Gas flow nebulization ( 2L/min 3L/min )
• Desolvatation temperature line ( 250°C 300°C)
• Heat Block temperature ( 400°C 500°C)
• Influence of nebulisation and Heat block temperature parameters
• Slight interaction between these two parameters
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350
2 3
Sign
al
Gas flow nebulization (L/min)
Effet
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400 500
Sign
al
Heat Block temeperature
Effet
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250 300
Sign
al
Temperature DL(°C)
Effet
Results
MMAF MMAE
ω < 0.25 min
Constant elution time: σ = 0.02 min 50 successive injections in plasma
Calibration
• Matrix : Nude mice plasma
• Plasma volume: 20µL doping with MMAE at various concentration
• Internal standard (MMAF) : 5µL at 50ng/mL -> concentration in 25µL of sample : 10 ng/mL
• Linearity range: • 3 ng/ml to 5µg/mL
• LOD : 1 ng/mL 1.2 fmol on column LOQ : 3 ng/mL in plasma sample 3.6 fmol on column
y = 0,0045x - 0,1038 R² = 0,9993
0
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25
0 1000 2000 3000 4000 5000 6000
Are
a ra
tio
Concentration (ng/mL)
MMAE Calibration internal standard Mice nude plasma
ADC spiking in nude plasma mice
Final concentration 100 µg/ml
Results Ex vivo Free MMAE
Incubation 37°C
Ex Vivo Free MMAE Assay
T0h, T8h, T24h T48h, T96h, T168h
ADC stability ELISA
Free MMAE TQ Quantification
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120
0,000 8 24 48 96 168
[AD
C] (
µg/m
l)
Timepoint (hour)
ADCETRIS in Nude plasma mouse 1 mouse2 mouse3 mouse4 mouse5
Results Ex vivo Free MMAE
Results Ex vivo Free MMAE
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900
-20 0 20 40 60 80 100 120 140 160 180
Conc
(ng/
mL)
Time Point Hour
Free MMAE ADCETRIS ex vivo
Results In Vivo Free MMAE
ADCETRIS injection 2 conditions
100µg (5mg/kg) 300 µg (15mg/kg)
Intravenous 100µl max injected/mouse
Different time point 5 minutes 24 hours 4 Days 7 Days
11 Days 14 Days
ELISA assay 5 minutes 24 hours 4 Days 7 Days
11 Days 14 Days
Free MMAE TQ 4 Days 7 Days
11 Days 14 Days
Results In Vivo Free MMAE
1,0
10,0
100,0
1000,0
0 50 100 150 200 250 300 350 400
[[A
DCE
TRIS
(µg/
ml)
Timepoint (hour)
PK ADCETRIS 100 vs 300 µg in Nude Elisa 300µg
Elisa 100µg
Results In Vivo Free MMAE
1,0
10,0
100,0
0 50 100 150 200 250 300 350 400
[[M
MA
E (n
g/m
l)
Timepoint (hour)
Free MMAE in Vivo 100 vs 300 µg in Nude
Triple Quad 100µg
Triple Quad 300µg
Results In Vivo Free MMAE
1,0
10,0
100,0
1000,0
0 50 100 150 200 250 300 350 400
[[A
DCE
TRIS
(µg/
ml)
MM
AE
(ng/
ml)
Timepoint (hour)
PK ADCETRIS 100µg vs 300µg VS Free MMAE in Vivo in Nude Elisa 300µg Elisa 100µg Triple Quad 100µg Triple Quad 300µg
Conclusion
• LC-MS/MS is an advantageous technique for Free MMAE quantification
• Fast sample preparation
• Good Sensitivity PK analysis
• Alternative technique to determine DAR