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

Periodic Reporting for period 1 - PRECALI (PREeclampsia risk stratification test: development of a suitable CALIbration framework for the analysis of a multiplex panel of endogenous compounds by LC-MS)

Reporting period: 2018-06-01 to 2020-05-31

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:
Delivering robust quantification data which can be fed to the risk stratification algorithm, taking into account: Long term reproducibility (e.g. change of reagent batches, calibrator materials) and Robustness – independent set-ups and laboratories should all generate data sets which are compatible with the risk stratification algorithm
Delivering robust quantification data in a format which can be accepted by key stakeholders

The project was successful in delivering the following outcomes which support the feasibility of commercialising a multi-metabolite biomarker based 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 achieved the following outcomes;
Preserved the (preterm) pre-eclampsia prediction as observed in the original biomarker discovery study.
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 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 work performed within the project lifetime was largely in line with the project as mapped out in the original proposal.

The intention was that appropriate Quantification concepts for endogenous Biomarker candidates would be considered and evaluated through stakeholder engagement and literature research - this was achieved.

Once appropriate Quantification concepts were identified, experimental work was carried out to test and establish the most suitable of those concepts in order to quantify four Biomarker candidates, as selected by METABOL in targeted metabolomic discovery studies.
Further work was carried out within the commercial laboratory (ISO 13485 accredited) to validate this concept, in line with the needs of the ongoing preeclampsia risk prediction product being developed. This work included a feasibility study with >1500 clinical subjects delivering valuable analytical figures of merit.

Key results of the assay development as outlined above were already presented by the researcher to the broader scientific community in the form of two posters. One was part of the MSACL-EU 2019 (Salzburg, Austria) and another had one originally been accepted for MSACL-US 2020 (Palm Springs, USA), but due to the pandemic it was presented in May 2020 at a virtual event instead, the Agilent Healthcare Heroes - Clinical Customer Appreciation Symposium. This was supported by Social Media activity from METABOL’s marketing department.

Further scientific dissemination, in the form of a scientific publication in a peer-reviewed journal, will be pursued beyond the timeline of the PRECALI project
Preeclampsia is a pregnancy related disorder affecting 5% of 1st time pregnancies. It remains a leading cause of maternal and perinatal morbidity and mortality resulting in the deaths each year of 70,000 mothers and 500,000 infants as a direct consequences of Preeclampsia. In the EU, with about 5.1 million births per annum (2015 data) , treating the women identified at high risk by PrePsia™ has the potential to prevent over 37,000 cases of Preterm PE and its associated severe outcomes, every year.
Knowing who is really at increased risk of preeclampsia and who is not, will provide valuable information to customise care of pregnant women. Currently clinicians have a limited set of tools to identify women at risk, which hampers early intervention. METABOL’s answer PrePsia™, a risk screening test for pre-eclampsia, will provide clinicians knowledge which can support personalised clinical intervention, ultimately leading to better pregnancy outcomes.
The reliable quantification of a pre-set panel of metabolites achieved in this project has enabled progression of PrePsia’s™ development in line with clinical needs and regulatory requirements. The inclusion of a highly sensitive and precise analytical technique such as SID-LC-MS/MS (Stable-Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry) enables the measurement of a significant combination of biomarkers in blood in one multiplex assay. Thus, the development of this assay constitutes a unique feature in the pre-eclampsia diagnostic field. PrePsia™ has the potential to revolutionise prenatal care globally by enabling healthcare providers to customize pregnancy care to a woman’s personal risk profile.