(T1130-06-39) Targeted Profiling of Lipid Mediators as Serum Biomarkers of Inflammation in a Case Study

Purpose: Lipid mediators are signaling biomolecules derived from polyunsaturated fatty acids (PUFA) linked with inflammatory response. The molecules are produced in circulation in response of extracellular stimuli. Since the molecules play key role in inflammation, disease, and resolution therefore they serve as biomarkers for severity of disease and therapeutic responses. The rationale of the study was to evaluate lipid mediators as biomarkers to monitor inflammatory responses of 400 subjects from a case study. The objective of the study was to perform targeted profiling of lipid mediators with a panel of 44 lipid mediators in 1200 serum samples from 400 subjects, comparing the levels of lipid mediators longitudinally post vaccination.

Methods: Workflow was developed for identification and relative quantitation of lipid mediators. The lipid mediators were identified using in-house mass spectral library containing 44 lipid mediator standards across different lipid classes with reversed phase liquid chromatography (LC) using Agilent 1290 UHPLC coupled with Sciex QTrap instrument using retention time and MRM transitions for each of the compounds. Bioanalytical assay was developed by utilizing calibration curve in reconstituting solvent as a surrogate matrix to determine limit of detection (LOD), lower limit of quantitation (LLOQ) and performance of the assay was determined. The lipid mediators were extracted from serum using solid phase extraction and analyzed with the bioanalytical assay developed. The MRM signal was used for semi-quantitative profiling. Limit of detection (LOD), lower limit of quantitation (LLOQ) and recovery in serum matrix were determined for the assay. The 1200 samples are split across 15 batches with pooled serum quality controls (QCs) used to determine batch to batch variability. The levels for lipid mediators for each of the subjects were compared between different timepoints to understand their role in inflammation resolution and classify subjects based on the severity of the inflammatory response.

Results: The LLOQ levels ranged between 50 pg/ml and 200 ng/ml with 66% of lipid mediators having LLOQ ≤ 150 pg/ml. The calibration curves were linear between LLOQ and 200 ng/ml. The recovery for over 80% of the lipid mediators in serum was determined to be between 80% and 120%. The assay is suitable for relative quantitation to evaluate percentage change in area ratio of lipid mediators (ratio of peak area of analyte of interest and peak area of corresponding internal standard) and statistical significance for each patient by comparing post treatment timepoints D1 baseline and D8. The results of one of the batches comprising of 27 subjects showed that levels of 17-HDHA linked with lower pain levels had percentage mean decline (23%) in D8 as compared to D1 baseline. The specialized pro-resolving lipid mediators with ability to resolve inflammation such as Resolvins: RvD1 (34%), RvD5 (37%) Protectin: PD1 (35%) and Maresin: Maresin1 (35%) in D8 as compared to D1 baseline.

Conclusion: LC-MS assay was developed to identify lipid mediator profile in subjects’ serum post vaccination that can be leveraged to identify inflammatory response in subjects associated with the vaccine and to support research of underlying pathways leading to discovery of new therapeutic targets.

Acknowledgements: Acknowledgements, disclaimers, funding, and other disclosure or conflict of interest statements: This study was funded by Moderna, Inc. AV, NB, LM, WY, DJ and GA are employees of Moderna, Inc. and may own stock/stock options in the company.