PREeclampsia risk stratification test: development of CALIbration framework for the analysis of a multiplex panel of endogenous Compounds by LC-MS.

Metabolomic Diagnostics (METABOL) is an innovative company which is developing tests for pre-eclampsia in pregnancy. One of the key challenges in the product development is establishing the optimal way to perform the quantification of the metabolite targets underpinning the preeclampsia test.
In the case of risk stratification products, the predictive metabolite biomarker profiles are merely a perturbation in the levels of a set of endogenous metabolites in the blood of pregnant women at risk of developing Preeclampsia. Because these predictive metabolites can be found in every person’s blood several technical challenges arise, for instance: the lack of readily available analyte-free matrix for the production of calibrators, or the fact that the internationally well-established LC-MS test validation framework for bioanalysis of xenobiotics is not readily applicable.The scope of the project therefore was to develop an appropriate quantification strategy for the product addressing the challenges above, and complying with the following pre-defined product development prerequisites:
1. Delivering robust quantification data which can be fed to the risk stratification algorithm, taking into account:
A) Long term reproducibility (e.g. change of reagent batches, calibrator materials)
B) Robustness – independent set-ups and laboratories should all generate data sets which are compatible with the risk stratification algorithm
2. Delivering robust quantification data in a format which can be accepted by key stakeholders
A) Regulatory Approval bodies
B) Clinical laboratory directors
C) Test prescribersThe project was successful in delivering the following high level outcomes which support the feasibility of commercialising a multi-metabolite biomarker based preeclampsia test:
• A newly developed LC-MS/MS assay which offers precise quantification for 4 pre-selected pre-eclampsia biomarker candidates in pregnant women’s plasma employing a seven point calibration curve and 3 levels of matrix-matched quality control levels (cf. to allow for batch QC as relevant for application of the assay in clinical laboratories).
• Absolute quantification of the endogenous molecules was achieved by implementation of state-of-the-art Isotope-dilution using matching Internal standards of high quality (no relevant crosstalk was detected).
• An assay design which took into consideration design input requirements regarding both the sample preparation protocol and the LC-MS/MS setup. The resulting assay was found to be sufficiently robust with low inter-instrument and inter-operator imprecision, both supporting the development into an IVD kit.
• In a medium scale feasibility study with clinical samples it was shown that the quantitative assay
o Preserved the (preterm) pre-eclampsia prediction as observed in the original biomarker discovery study.
o Was of such quality that batch acceptance criteria based on international guidelines were met
• Furthermore, the measuring interval was established for EDTA plasma samples obtained at 11-13 weeks of pregnancy samples, in a proof of principle test involving >1500 clinical samples.
In addition to the further development of the researcher’s scientific skills and expertise, the Marie Curie fellow developed significant business skills during the lifetime of the project, which will be instrumental in her career progression. Notably, the researcher successfully studied for her Lean Six Sigma Yellow Belt qualification and implemented a successful continuous improvement project within the company’s product development utilising those techniques. Moreover, the researcher was further exposed to a range of project management techniques, team building, risk management, supervision, mentoring etc. within METABOL.
Also of significance was the researcher’s exposure to and engagement with a number of the aspects of the complex pathway to delivering an IVD to the commercial market place, including the work in a regulated environment (ISO13485), and engagement with relevant stakeholders. The opportunity to build understanding of this commercially important product development framework provided the Marie Curie fellow with an important professional development step-up.