(M1030-10-67) Determination of the Concentration of the Drug MG1113 in Human Sodium Citrate Plasma Using a Sandwich Electro Chemiluminescent Immunoassay (ECLIA)

Purpose: In this study, we developed a sandwich electro chemiluminescent immunoassay (ECLIA) method to quantitate MG1113 concentration in human sodium citrate plasma to support clinical analysis.

Methods: MG1113 concentration is determined from 90 µL human sodium citrate plasma that has been diluted 1.6-fold in 330 mM glacial acetic acid and subsequently neutralized, and then analyzed using a sandwich electro chemiluminescent immunoassay (ECLIA). For this assay, standards, quality controls (QC), and stability QC samples were prepared in pooled human sodium citrate plasma. The samples are loaded onto the Meso Scale Discovery (MSD) multi-array plate that was previously coated with recombinant human Tissue Factor Pathway Inhibitor (TFPI) protein and blocked. The plate is incubated to allow the MG1113 present in samples to bind, washed, and biotinylated detection antibody added to the plate to detect captured MG1113. After further incubation and washing, sulfo-tag labelled streptavidin is added and allowed to incubate. After the final incubation, the plate is washed and 1X MSD Read Buffer T is applied. The signal is measured using an MSD Quick Plex SQ120 plate reader, which applied a current to the MSD plate-based electrodes in each well. The resulting oxidation-reduction reaction between ruthenium (sulfo-tag) and tripropylamine (TPA), contained in the MSD read buffer, emits light, which is measured by the plate reader. The intensity of light emitted, measured as electro chemiluminescence units (ECLUs), is proportional to the concentration of MG1113 present in stability QC samples. The ECLUs obtained from the calibration standards are fitted using a 4-parameter logistic (4-PL) fit equation with 1/y² weighting to calculate the MG1113 concentrations in the samples. Method parameters, including sensitivity, precision, bias, selectivity, dilution linearity, and stability, were evaluated and are presented in this study.

Results: Data acquisition was carried out using MSD methodical Mind^TM version 1.0.38. The resulting raw data generated from the MSD software was imported into Watson LIMS version 7.6.1. Statistical parameters such as means, standard deviations, and coefficient of variation and relative error were exported from Watson LIMS. The method for MG1113 showed excellent linearity using a 1/concentration² weighting at a concentration range of 10 - 640 ng/mL and a mean correlation of coefficient of 0.9917. For LLOQ samples, the intra-assay coefficient of variation (CV) ranged from 0.8% to 13.3%, the relative error (RE) ranged from -20.8% to 24.0%; the inter-assay CV was 16.8%, the RE was -3.8%. For ULOQ samples, the intra-assay CV ranged from 1.5% to 9.6%, the RE ranged from -15.6% to 12.8%; the inter-assay CV was 11.0%, the RE was -5.6%. For LQC, MQC, and HQC samples, the intra-assay CV ranged from 0.6% to 8.0%, the RE ranged from -6.0% to 10.4%; the inter-assay CV ranged from 4.1% to 6.4%, the RE ranged from -1.6% to 2.9%. Selectivity demonstrated no significant interference. Dilutional linearity samples demonstrated means within 20% of nominal for all dilutions tested and no prozone effect was observed. RE for Bench-top stability samples stored at ambient temperature for 18 hours ranged from -5.0% to 9.2% for samples stored at -20°C and -10.5% to 4.9% for samples stored at -70°C. RE for samples undergoing four freeze-thaw cycles ranging from -9.5% to 6.3% for samples stored at -20°C and from -7.5% to 6.4% for samples stored at -70°C. Long term storage stability (LTSS) was established for 35 days, RE ranged from -6.8% to 0.4% for samples stored at -20°C and from -7.2% to -2.7% for samples stored at -70°C.

Conclusion: A sandwich electro chemiluminescent immunoassay methods for the quantitation of MG1113 in human sodium citrate plasma by Meso Scale Discovery has been established. These procedures allow for rapid translation of study samples into data to support clinical analysis.

References: MBL Method Validation Report “a Ligand Binding Assay for the Quantitative Determination of MG1113 in Human Sodium Citrate Plasma by Electrochemiluminescence on the Meso Scale Discovery Quick Plex SQ120”, 2023