Bevacizumab

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Jerome Vialaret1, Célia Puginier1, Laurent Tiers1, Sophie Broutin2, Angelo Paci2, Laurent Pelletier3, Sylvain ... Bevacizumab (Avastin®, Roche-Genentech) is a.

New quantification method of bevacizumab using a protein A immuno-enrichment and LC-QqQ analysis Jerome Vialaret1, Célia Puginier1, Laurent Tiers1, Sophie Broutin2, Angelo Paci2, Laurent Pelletier3, Sylvain Lehmann1 and Christophe Hirtz1 1: Biochemistry Laboratory – Platefome de protéomique Clinique (PPC); IRMB, CHRU Montpellier, France 2: Institut Gustave Roussy, Service de Pharmacologie et UMS CNRS 3655 & INSERM US23 AMMICa, Laboratoire de Pharmacologie et d’Analyse (LPA), Villejuif, France 3: Grenoble Institut des Neurosciences (GIN) INSERM U836, Safra Chemin Fortuné Ferrini, La Tronche, France

For research use only. Not for use in diagnostic procedures.

Biochemistry Lab. Clinical diagnosis activities -

Biological diagnosis of Dementia (Alzheimer Disease, Prion diseases..)

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Biological diagnosis of Muscular dystrophy

Clinical Research activities -

Biomarker discovery programs (coordinator and partner)

Biobanking activities

Memory Research Resources center Department of Neurology, Montpellier University Hospital

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(internal and external) with up to 10000 samples

(Blood, plasma, CSF, saliva, DBS)

For research use only. Not for use in diagnostic procedures.

Biochemistry Lab.

Sylvain Lehmann Head of the LBPC

Christophe Hirtz Head of the PPC

Jerome Vialaret 

MS Specialist For research use only. Not for use in diagnostic procedures.

MS assays on Neurodegenerative disorders Amyloid Peptides

TAU proteins (proteoforms)

PMID: 26923020; 26742856; 26388715; 2571932; 26707914; 271165941; DOI: 10.1016 For research use only. Not for use in diagnostic procedures.

ApoE Phenotyping

MS assays on Neurodegenerative disorders Amyloid Peptides

TAU proteins (proteoforms)

Hepcidin-25

Orexin A

PMID: 26923020; 26742856; 26388715; 2571932; 26707914; 271165941; DOI: 10.1016 For research use only. Not for use in diagnostic procedures.

ApoE Phenotyping

Bevacizumab / Avastin - VEGF (Vascular endothelial growth factor) is

overexpressed in most human tumors and that increased VEGF expression is associated with tumor progression and/or risk of cancer recurrence (Ternant, 2010).

- Bevacizumab (Avastin®, Roche-Genentech) is a humanized monoclonal immunoglobulin G1 (IgG1) antibody that specifically binds circulating vascular endothelial growth factor (VEGF). It is used to limit tumor vascularization.

Bevacizumab is used in combination with standard chemotherapy, and is approved in breast cancer (cf. Han, AAPS journal, 2014) metastatic colorectal cancer non-small cell lung cancer renal cell carcinoma ovarian cancer glioblastoma

For research use only. Not for use in diagnostic procedures.

Pharmacokinetics mAbs: - Long half-life: close to 21 days - The steady state conditions are generally reached after 15 weeks of a mAb administration at the usual 2-4 week intervals (Azanza, 2014). Avastin: - Mean half-life: 20 days (Roche) - Individual differences: 11 to 50 days

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Patient administration must be adjusted Standard Administration protocol: 10mg/kg every 2 weeks

Normal clearance

High clearance

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(Ternant, 2010) (Widemer, 2014)

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Lack of antibody after one week

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Low clearance

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Back to the baseline just before the second dose

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2 week

Excess of circulating antibody before the second dose

For research use only. Not for use in diagnostic procedures.

Pharmacokinetics inter-individual variability factors

k0

Gender

kin

Body Weight Serum Albumin

k12

Phosphatase alkaline Concomitant chemotherapies

AVA + VEGF

AVA V2

Kss

k21

AVA-VEGF

ke2

V1

SGOT Tumor burden VEGF-A polymorphism

ke1 kout

Height Receptor FcRn (Ternant, 2016) (Ternant, 2010) (Widemer, 2014) (Lu, 2008)

Schematic representation of Avastin (AVA)pharmacokinetics based on two-compartment model. V1 : Distribution volume in central compartment; V2:Distribution volume in peripheric compartment; k0 : enter constant; k12 , k21 intercompartmental constant ; ke1 : elimination constant of free bevacizumab ke2 : elimination constant of complex ; kin : production constant of VEGF ; kout : elimination constant of VEGF ; Kss: equilibrium constant at steady-state For research use only. Not for use in diagnostic procedures.

Pharmacokinetics - Treatment 10mg/kg every 2 weeks 1 cycle

10mg/kg every 2 weeks 7 cycles

Theoretical Avastin concentration in a 70kg patient with treatment by 7 repeat administrations of 10 mg/kg every two weeks

Aim of quantitative assay

MS quantitative assay After 7 weeks of treatment

[Avastin]Blood high level could increase the occurrence of side effects (hemorrhage, phlebitis)

Therapeutic range Low level could compromise treatment efficacy

For research use only. Not for use in diagnostic procedures.

Case Study Brain cancer (n=15) with different treatment points -> 94 samples (duplicate on analysis) Avastin was intravenously infused at 10 mg/kg of body weight every two weeks. Blood samples were taken just before Avastin infusion. 10uL of serum was used to perform the assay 100µL of PBS 1X

10µl of acidified serum

Sample preparation

Protein A Immunoenrichment SILUMAb

Trypsin digestion

Peptide Clean-up C18

LC-MRM

For research use only. Not for use in diagnostic procedures.

Avastin Structure

3 peptides from the heavy chain, and one from the light chain were monitored For research use only. Not for use in diagnostic procedures.

Peptide selection Light Chain: VLIYFTSSLHSGVPSR

Heavy Chain: GLEWVGWINTYTGEPTYAADFK RFTFSLDTSK STAYLQMNSLR

ConSurf software

Peptides were located on the variable part of the antibody -> Proteotypic For research use only. Not for use in diagnostic procedures.

1- First step 100µL of PBS 1X

Sample preparation

10µl of acidified serum

SILUMAb 80ng

3 peptides well detected on our workflow. DTLMISR exhibit a lower CV and will use to normalize experiments For research use only. Not for use in diagnostic procedures.

2- Protein-A Capture

Antibodies capture

Tips ProtA

Prot-A

≈50µm

BRAVO Agilent

For research use only. Not for use in diagnostic procedures.

2- Protein-A Capture

Decomplexification of the sample by prot-A capture was very efficient For research use only. Not for use in diagnostic procedures.

3- Analytical method

30min LC-MRM run

Reversed-phase column (RRHD Eclipse Plus C18, 2.1x150mm, 1.8um) at 400µL/min. 30 min multi-step gradient (B: 2.7% at 0min; 9.9% at 2min; 17.1% at 15min; 26.1% at 22min; 40.5% at 25min; then flush and re-equilibration).

Peptides analysis were carried out on a QqQ MS system (6490, Agilent technologies), equipped with an Agilent Jet-Stream ESI interface and performed in positive ion mode. The MS operated in dynamic MRM with a retention time window of 3 minutes and a maximum cycle time fixed at 800ms. For research use only. Not for use in diagnostic procedures.

Analytical validation Blank serum was used as matrix. + 80 ng SILUMAb + 0; 10; 50; 100; 250; 500; 750 and 1000 ug/mL of Avastin Relative Responses

FTFSLDTSK.light - 9 Levels, 8 Levels Used, 55 Points, 48 Points Used, 0 QCs x10 1 y = 4.366238 * x + 0.001076 R^2 = 0.97286127 Type:Linear, Origin:Ignore, Weight:1/y 6

FTSLDTSK y=4.3662x+0.0011 R2: 0.9729 LOB: 1.18 ug/mL LOD: 1.94 ug/mL LOQ: 5.86 ug/mL

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Figure: Calibration curves of FTSLDTSK 6 repetitions; 8 points calibration curves (0; 10; 50; 100; 250; 500; 750 and 1000 ug/mL)

All the peptides shown a nice linear response For research use only. Not for use in diagnostic procedures.

Analytical validation Parameters checked to the validation: - Repeatability (6x), Reproducibility (4 days) - Limit of Blank (LOB), Limit of Detection (LOD), Limit of Quantification (LOQ), Accuracy - Carry-over; interferences; matrix effect tests - Workflow recovery; dilution integrity - Autosampler stability (24 hours)

STAYLQMNSLR

STAYLQMoxNSLR 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

Range (ug/mL)

R2 LOQ (ug/mL) CV rt (%)

RFTFSLDTSK

CV concentration (%) Accuracy (%) Reproducibility CV (%) Reproducibility Accuracy (%) Dilution Integrity Stability at 24 hour

GLEWVGWINTYTGEPTYAADF K

VLIYFTSSLHSGVPSR

FTFSLDTSK

Analytical validation Parameters checked to the validation: - Repeatability (6x), Reproducibility (4 days) - Limit of Blank (LOB), Limit of Detection (LOD), Limit of Quantification (LOQ), Accuracy - Carry-over; interferences; matrix effect tests - Workflow recovery; dilution integrity - Autosampler stability (24 hours)

STAYLQMNSLR

STAYLQMoxNSLR 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

Range (ug/mL)

R2 LOQ (ug/mL) CV rt (%)

RFTFSLDTSK

CV concentration (%) Accuracy (%) Reproducibility CV (%) Reproducibility Accuracy (%) Dilution Integrity Stability at 24 hour

GLEWVGWINTYTGEPTYAADF K

VLIYFTSSLHSGVPSR

FTFSLDTSK

Analytical validation Parameters checked to the validation: - Repeatability (6x), Reproducibility (4 days) - Limit of Blank (LOB), Limit of Detection (LOD), Limit of Quantification (LOQ), Accuracy - Carry-over; interferences; matrix effect tests - Workflow recovery; dilution integrity - Autosampler stability (24 hours)

STAYLQMNSLR

STAYLQMoxNSLR 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

Range (ug/mL)

R2 LOQ (ug/mL) CV rt (%)

RFTFSLDTSK

CV concentration (%) Accuracy (%) Reproducibility CV (%) Reproducibility Accuracy (%) Dilution Integrity Stability at 24 hour

GLEWVGWINTYTGEPTYAADF K

VLIYFTSSLHSGVPSR

FTFSLDTSK

After analytical validation, 4 peptides was selected (better analytical properties)

Quantitative assay Jia Wan et al. 2012

Heavy Chain: GLEWVGWINTYTGEPTYAADFK - - - R-FTFSLDTSK-STAYLQMNSLR Heavy Chain (deamidation): GLEWVGWINTYTGEPTYAADFK - - - R-FTFSLDTSK-STAYLQMNSLR Heavy Chain (Miss cleavage): GLEWVGWINTYTGEPTYAADFK - - - R-FTFSLDTSK-STAYLQMNSLR Heavy Chain (Met oxidation): GLEWVGWINTYTGEPTYAADFK - - - R-FTFSLDTSK-STAYLQMNSLR Heavy Chain (methylation): GLEWVGWINTYTGEPTYAADFK - - - R-FTFSLDTSK-STAYLQMNSLR Heavy Chain (nitrotyrosine): GLEWVGWINTYTGEPTYAADFK - - - R-FTFSLDTSK-STAYLQMNSLR

GLEWVGWINTYTGEPTYAADFK RFTFSLDTSK STAYLQMNSLR STAYLQMoxNSLR VLIYFTSSLHSGVPSR Correlation coefficient

FTFSLDTSK

Significance Level P n

0.919