Eloi Haudebourg1, Yvan Eb-Levadoux1, Catherine Pech1, François Autelitano1, Berenice Rotty1, François Lantreibecq1, Yannick Cogne1, Navratan Bagwan1, Alessia Cavaliere2, Rossella Cardin2, Alberto Vezzelli2, Alessandro Greco2,
Philipp Ellinger3, Wiebke Afhueppe3, Karoline Droebner3, Winfried Wunderlich4
1 Evotec (France) SAS Toulouse, France
2 Aptuit (Verona) Srl, an Evotec Company, Verona, Italy)
3 Bayer AG Pharmaceuticals Research, Berlin, Germany
4 Evotec SE, Goettingen, Germany
Introduction: While proteomics tools are increasingly used in the early steps of the drug development journey, its use remains limited to exploratory endpoints in clinical phase. Successful MS methods used in the exploratory and preclinical phases are adapted to antibody-based assays. For the present study, no antibody of the target engagement biomarker could be validated for animals in preclinical phase. An MS-based protein quantification method was therefore developed in Research Use Only (RUO) and ultimately transferred to a Good Clinical Practice (GCP) environment for validation and application to support preclinical development and clinical trial Phase 1 as a primary endpoint.
Methods: First, a high purity Stable Isotope Labelled (SIL) protein was produced in HEK293 cells cultured in SILAC medium and purified (anti-poly-histidine followed by IMAC and SEC). Second, a targeted MS assay was developed for the absolute quantification of biomarker of interest. Briefly 2 µL of plasma was processed on an iST kit (Preomics), peptides were injected using a microflow LC (M Class, Waters) on a C18-CSH column (Acquity, Waters) and quantified in MRM mode on a triple quadrupole (6500+, Sciex).
Preliminary data: Acceptable protein purity (> 80 % based on MS intensity) and excellent heavy label incorporation (>98 %) was archived. A targeted proteomics assay was first developed in RUO and transferred to a GCP facility where the method was validated according to FDA validation of bioanalytical methods for Industry and ICH-M10 guidelines. Specifically, the method validation included the assessment of precision and accuracy of the assay, surrogate matrix equivalence, lower limit of detection (100 ng/ml), concentration range, short- and long-term stability of the samples.
The validated, targeted MS method is routinely used to quantify a target engagement biomarker in a currently ongoing Phase 1 clinical trial.
Novel aspects: First time for the use of MS-based targeted proteomics to quantify proteins in clinical samples as primary endpoint.