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.

_{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}