MSACL 2016 EU Abstract

New Method of Bevacizumab Dosage, a Monoclonal Antibody, Thanks to a Protein A Immunoenrichment Following by a LC-QqQ Analysis

Jerome Vialaret (Presenter)
CHU Montpellier

Bio: Jerome Vialaret was born in 1984 and studied Organic Chemistry at Montpellier University (Master degree in 2007). Specialization in proteomic was made through jobs on Pierre Fabre Laboratories (Oncology center, France), EPFL (Lausanne, Switzerland), INRA (Montpellier, France) until Montpellier hospital. After huge experience in large scale proteomic he focused on the development of protein quantification using targeted mass spectrometry in a clinical environment. He is in charge of 3 LCMS systems, 2 triple quadrupole (6490 Agilent T.; 8060 Shimadzu) and one HRMS system (Impact II, Bruker D.). Targeted MS assays were developed and validated on field of neurodegenerative diseases (TAU protein, Abeta peptides, ApoE phenotyping, hepcidin-25 and orexin-A. In parallel, he performed a multiplex quantitative assay on 76 protein biomarkers apply on plasma, CSF, urine, saliva and DBS.

Authorship: Jerome Vialaret (1), Célia Puginier (1), Laurent Tiers (1), Sophie Broutin (2), Angelo Paci (2) , Sylvain Lehmann (1) and Christophe Hirtz (1)
(1) Laboratoire de Biochimie et Protéomique Clinique, IRMB, CHU de Montpellier, Montpellier, France; (2) Institut Gustave Roussy, Dep de Biologie et Pathologie Médicales - Service de Pharmacologie et UMS CNRS 3655 & INSERM US23 AMMICa

Short Abstract

Monoclonal antibody (mAb) is the first class of biotechnological products followed by recombinant proteins and vaccines. Bevacizumab (AVASTIN) product by Roche, is able to neutralize Vascular Endothelial Growth factor (VEGF), a regulator of angiogenesis and surexpressed in a lot of human tumors. By combining a step of Protein-A capture and a LC-MRM analysis, Bevacizumab could be quantify on patient serum during cure (cycle 1, 3, 5, 7, 9, 11 and 13). This analytical approach was fully validated (SiluMab was used as internal standard) and compare to home-made elisa test.

Long Abstract


Since advents of biotechnologies, biological medicine takes an important part of pharmaceutical market particularly in oncology, auto-immune diseases. Growth of this market will continue during the next years [1]. Monoclonal antibody (mAb) is the first class of biotechnological products followed by recombinant proteins and vaccines[2]. AVASTIN® product by Roche, is a mAb produced from recombinant DNA of Chinese hamster ovary cells[3]. Bevacizumab is a recombinant humanized immunoglobulin gamma 1. This mAb is able to neutralize Vascular Endothelial Growth factor (VEGF), a regulator of angiogenesis and surexpressed in a lot of human tumors.

The medicine is involved to be more personalized in different goals. Two of these were to be more efficient against pathology, and to limit side effects. Monoclonal antibodies are treatments associated with serious side effects as frequent affections of cardiovascular, neurological, immune system disorders for example. PK/PD studies can explain the relation between drug concentration and pharmacodynamics response to establish optimal dosage in order to reduce painfulness and increase efficacy of treatment.


A prospective observational study will be submitted to the local CPP in order to realize a PK/PD analysis on a blood collection from metastatic breast cancer patients treated at Gustave Roussy cancer center with Bevacizumab (10 mg/kg every two weeks - infusion) and in combination with paclitaxel (collaboration with Dr Mir, Gustave Roussy). Patients (n=15) will provide a written informed consent for participating in this study. Blood samples (serum collection tube – 5 mL) will be collected just before and two hours after Bevacizumab infusion at cycle 1, 3, 5, 7, 9, 11 and 13 in order to measure bevacizumab serum concentrations.

Before LCMS analysis, a step of Protein-A capture was introduced to improve the sensitivity of the method. Thanks to AssayMap BRAVO, protein-A tips were available. Selected cartridges contain a recombinant Staphylococcus aureus protein A linked to a chromatography column. Protein A has a high selectivity to Fc fragment of IgG. This recombinant protein adsorbs bevacizumab and others Igs with a lower affinity[11]. Samples were loaded that allowed contact between serum and protein A. Flow through and two washes were trashed. Finally, samples were eluted with HCl 12 nM, NaCl 100mM to stop interact of Fc with protein A and recover proteins of interest in elution.

Eluted proteins were digested 24 hours at 37°C with trypsin. Peptides were cleaned on C18 cartridges, dried, resuspended with water containing 3% acetonitrile and 0.1% formic acid, before LCMS injection.

Mass spectrometry analysis was performed on 30 minutes thanks to an LC column of 2.1x150mm (1.8um) at 400µL/min. Triple quadrupole system performed a quantification on 4 proteotypic peptides (one for light chain and 3 for the heavy chain). Accurate quantification was access by performing calibration curve on matrix (blank serum) and with normalization by external standard (silumab) spiked on raw material.

Preliminary data

No available method was currently use in clinical environment to quantify AVASTIN® on serum. Automated Protein-A capture/protein digestion follows by LC-MRM assay was developed and validate to perform it. Mass spectrometry gives access to very specific data by double filtering on MS level. Specificity, matrix effect and carry-over of the analysis were evaluated during the analytical method validation. Analytical performances were also evaluated without compromise: LOB, LOD, LOQ, reproducibility, repeatability, trueness and precision.

The 4 peptides were under the analytical limit of acceptance.

A clinical range of 10 to 500 ug/mL was established on patient before bevacizumab infusion cycles.

Novel aspect

LC-MRM method was qualified to perform a quantitative assay on bevacizumab from patient sera.

Statistical analysis on many patients, in combination to biological data, could give access to personalized medicine in context of metastatic breast cancer.

References & Acknowledgements:

Financial Disclosure

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