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 Monoclonal antibody
ntibody rug onjugate Anatomy of an ADC
ntibody Linker rug T T onjugate T T Toxin Brentuximab-Vedotin
cAC10 Chimeric IgG1 ADCETRIS ntibody rug onjugate
maleimidecaproyl Dipeptide p-aminobenzylcarbamate Monomethyl auristatin E Valine Citrulline 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
MMAE Chemical structure Dolastatin 10 Chemical structure
• High toxicity Wedge sea hare • Blocks tubulin polymerization ADC Mechanism 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
Liquid MMAE Mass Chromatography Spectrometry Extraction Separation Analysis Methods
• MMAE protocol extraction SPE (MMAE High toxicity)
20µl plasma 5µl internal Protein standard Precipitation MMAF spiking (50µL MeOH)
Recovery LC-MS/MS supernatant Centrifugation Analysis (40 µL) Triple quadrupole (in 80µL H 2O/MeOH) Methods
• Chromatography • Kinetex XB-C18 Phenomenex UHPLC Column
• Eluent : H 2O = 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
Gradient
100 90 80 70 60 50
40 % Phase B
% organic phase 30 20 10 0 0 1 4 5,5 7 7,1 8 Minutes Methods
• Mass spectrometry triple quadrupole • Triple quadrupole using SRM (Single Reaction Monitoring)
Q1 Q2 Q3 Detection igerato monitoring reaction Single
SIM Fragmentation SIM
• Increase the signal to noise ratio 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 MMAF
Transition Dwell time (ms) Q1 PreBias (V) Collision Energy (V) Q3 PreBias (V) Transition Dwell time (ms) Q1 PreBias (V) Collision Energy (V) Q3 PreBias (V)
718,5->686,3 100 -28 -31 -34 732,5->700,3 100 -28 -29 -26
718,5->152,0 100 -28 -36 -29 732,5->170,1 100 -28 -46 -30 Optimization
Mobile phase optimization
MMAE signal response optimization MMAF signal response 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
350 350 350 300 300 300 250 250 250 200 200 200 Signal
Signal Signal 150 150 Effet 150 Effet Effet 100 100 100 50 50 50 0 0 0 2 3 250 300 400 500 Gas flow nebulization (L/min) Temperature DL(°C) Heat Block temeperature 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
MMAE Calibration internal standard Mice nude plasma 25 y = 0,0045x - 0,1038
20 R² = 0,9993
15
Area ratio 10
5
0 0 1000 2000 3000 4000 5000 6000 Concentration (ng/mL) Results Ex vivo Free MMAE
Ex Vivo Free MMAE Assay
ADC stability ELISA
Incubation 37 °C
ADC spiking in nude plasma mice Free MMAE TQ Final concentration 100 µg/ml T0h, T8h, T24h Quantification T48h, T96h, T168h Results Ex vivo Free MMAE
ADCETRIS in Nude plasma 120 mouse 1 mouse 115 2 mouse 3 110 mouse 4 mouse 105 5
100
95 [ADC] (µg/ml) [ADC]
90
85
80 0,000 8 24 48 96 168 Timepoint (hour) Results Ex vivo Free MMAE
Free MMAE ADCETRIS ex vivo
900
800
700
600
500
400 Conc(ng/mL)
300
200
100
0 -20 0 20 40 60 80 100 120 140 160 180 Time Point Hour Results In Vivo Free MMAE
Shah et al. J Pharmacokinet Pharmacodyn (2012) Results In Vivo Free MMAE
Kaur et al. Bioanalysis (2013) Results In Vivo Free MMAE
ELISA assay 5 minutes 24 hours 4 Days 7 Days 11 Days 14 Days ADCETRIS injection 2 conditions Different time point 100µg (5mg/kg) 5 minutes Free MMAE TQ 300 µg (15mg/kg) 24 hours 4 Days Intravenous 4 Days 7 Days 100µl max injected/mouse 7 Days 11 Days 11 Days 14 Days 14 Days Results In Vivo Free MMAE
1000,0 PK ADCETRIS 100 vs 300 µg in Nude Elisa 300µg Elisa 100µg
100,0 [[ADCETRIS [[ADCETRIS (µg/ml)
10,0
1,0 0 50 100 150 200 250 300 350 400 Timepoint (hour) Results In Vivo Free MMAE
100,0 Free MMAE in Vivo 100 vs 300 µg in Nude Triple Quad 100µg
Triple Quad 300µg
10,0 [[MMAE (ng/ml)
1,0 0 50 100 150 200 250 300 350 400 Timepoint (hour) Results In Vivo Free MMAE
1000,0 PK ADCETRIS 100µg vs 300µg VS Free MMAE in Vivo in Nude Elisa 300µg Elisa 100µg Triple Quad 100µg Triple Quad 300µg
100,0
10,0 [[ADCETRIS [[ADCETRIS (µg/ml) MMAE (ng/ml)
1,0 0 50 100 150 200 250 300 350 400 Timepoint (hour) Conclusion
• LC-MS/MS is an advantageous technique for Free MMAE quantification • Fast sample preparation • Good Sensitivity PK analysis • Alternative technique to determine DAR Acknowledgements
• Mikael Levi • Daniel Lafitte • Stéphane Moreau • Christopher Nuccio • Claude Villard • François Gray • Rima Aït-Belkacem • Thérèse Schembri T T • Lina Sellami Angélique Boedec • T T • Agnès Represa • Hélène Rispaud • Sandra Savard-Chambard
T T