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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
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Overview
• Introduction
• Antibody Drug Conjugate
• General
• ADC Brentuximab-Vedotin
• Monomethyl Auristatin E
• Equipment
• Methods
• Results
• Conclusion
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Monoclonal antibody
ntibody
onjugaterug
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ntibody
onjugate
rug Linker
T
T
T
T
Toxin
Anatomy of an ADC
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ntibody
onjugate
rug
ADCETRIS
Brentuximab -Vedotin
cAC10
Chimeric IgG1
maleimidecaproyl Dipeptide
Valine
Citrulline
p-aminobenzylcarbamate Monomethyl auristatin E
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Monomethyl auristatin E
• The auristatin E synthetic analog of the natural product dolastatin 10
• Dolastatin 10 was originally isolated from the Indian Ocean seahare, Dolabella auricularia
• High toxicity
• Blocks tubulin polymerization
MMAE Chemical structure Dolastatin 10 Chemical structure
Wedge sea hare
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ADC Mechanism
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LC-MS/MS system
• SHIMADZU 8040 LC-MS/MS
• Liquid Chromatography UHPLCNexera SHIMADZU
• Electrospray interface
• Triple quadrupole
• Speed : up to 555 MRM/sec
• Sensitivity : ≈ 1 fmol(Réserpine M=608.7 S/N 10000)
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Methods
MMAE
Extraction
Mass Spectrometry
Analysis
Liquid
Chromatography
Separation
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Methods
• MMAE protocol extraction
20µl plasma5µl internal
standard
MMAF spiking
Protein
Precipitation(50µL MeOH)
Centrifugation
Recoverysupernatant
(40 µL)(in 80µL H2O/MeOH)
SPE (MMAE High toxicity)
LC-MS/MS
Analysis
Triple quadrupole
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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
0
10
20
30
40
50
60
70
80
90
100
0 1 4 5,5 7 7,1 8
% o
rga
nic
ph
ase
Minutes
Gradient
% Phase B
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Methods
• Mass spectrometry triple quadrupole
• Triple quadrupole using SRM (Single Reaction Monitoring)
Q1 Q2 Q3
SIM Fragmentation SIM
Sin
gle
rea
ction
mo
nito
ring
• Increase the signal to noise ratio
De
tectio
n
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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
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Optimization
MMAE signal response optimization MMAF signal response optimization
Mobile phase optimization
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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
0
50
100
150
200
250
300
350
2 3
Sig
na
l
Gas flow nebulization (L/min)
Effet
0
50
100
150
200
250
300
350
400 500
Sig
na
l
Heat Block temeperature
Effet
0
50
100
150
200
250
300
350
250 300
Sig
na
l
Temperature DL(°C)
Effet
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Results
MMAF MMAE
ω < 0.25 min
Constant elution time: σ = 0.02 min 50 successive injections in plasma
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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
5
10
15
20
25
0 1000 2000 3000 4000 5000 6000
Are
a r
ati
o
Concentration (ng/mL)
MMAE Calibration internal standard Mice nude plasma
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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, T24hT48h, T96h, T168h
ADC stability
ELISA
Free MMAE TQ
Quantification
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80
85
90
95
100
105
110
115
120
0,000 8 24 48 96 168
[AD
C] (
µg/m
l)
Timepoint (hour)
ADCETRIS in Nude plasma mouse1mouse2mouse3mouse4mouse5
Results Ex vivo Free MMAE
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Results Ex vivo Free MMAE
0
100
200
300
400
500
600
700
800
900
-20 0 20 40 60 80 100 120 140 160 180
Co
nc
(ng
/mL)
Time Point Hour
Free MMAE ADCETRIS ex vivo
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Results In Vivo Free MMAE
Shah et al. J Pharmacokinet Pharmacodyn (2012)
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Results In Vivo Free MMAE
Kaur et al. Bioanalysis (2013)
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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 assay5 minutes
24 hours
4 Days
7 Days
11 Days
14 Days
Free MMAE TQ4 Days
7 Days
11 Days
14 Days
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Results In Vivo Free MMAE
1,0
10,0
100,0
1000,0
0 50 100 150 200 250 300 350 400
[[A
DC
ET
RIS
(µ
g/m
l)
Timepoint (hour)
PK ADCETRIS 100 vs 300 µg in Nude
Elisa 300µg
Elisa 100µg
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Results In Vivo Free MMAE
1,0
10,0
100,0
0 50 100 150 200 250 300 350 400
[[M
MA
E (
ng
/ml)
Timepoint (hour)
Free MMAE in Vivo 100 vs 300 µg in Nude
Triple Quad 100µg
Triple Quad 300µg
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Results In Vivo Free MMAE
1,0
10,0
100,0
1000,0
0 50 100 150 200 250 300 350 400
[[A
DC
ET
RIS
(µ
g/m
l)
MM
AE
(n
g/m
l)
Timepoint (hour)
PK ADCETRIS 100µg vs 300µg VS Free MMAE in Vivo in NudeElisa 300µg
Elisa 100µg
Triple Quad 100µg
Triple Quad 300µg
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Conclusion
• LC-MS/MS is an advantageous technique for Free MMAE quantification
• Fast sample preparation
• Good Sensitivity PK analysis
• Alternative technique to determine DAR
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TT
Acknowledgements
• Daniel Lafitte
• Christopher Nuccio
• Claude Villard
• François Gray
• Rima Aït-Belkacem
• Thérèse Schembri
• Lina Sellami
• Mikael Levi
• Stéphane Moreau
• Angélique Boedec
• Agnès Represa
• Hélène Rispaud
• Sandra Savard-Chambard
T
TT
T