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

onjugaterug

ntibody

onjugate

rug Linker

T

T

T

T

Toxin

Anatomy of an ADC

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 seahare, Dolabella auricularia

• High toxicity

• Blocks tubulin polymerization

MMAE Chemical structure Dolastatin 10 Chemical structure

Wedge sea hare

ADC Mechanism

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)

Methods

MMAE

Extraction

Mass Spectrometry

Analysis

Liquid

Chromatography

Separation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Conclusion

• LC-MS/MS is an advantageous technique for Free MMAE quantification

• Fast sample preparation

• Good Sensitivity PK analysis

• Alternative technique to determine DAR

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