for the treatment of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

for the treatment of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1?3 The antibody cocktail includes two humanized IgG1 monoclonal antibodies (REGN10933 and REGN10987), which are designed to target nonoverlapping epitopes around the SARS-CoV-2 spike protein, thereby blocking the interaction of SARS-CoV-2 computer virus with human ACE21 and preventing viral escape due to rapid genetic mutation of the virus.4 A recent clinical study has shown that REGEN-COV therapy can reduce viral weight and improve symptoms for nonhospitalized COVID-19 patients, especially those who were seronegative or HIF1A had high viral loads at baseline.3 Based around the promising results from the clinical investigation, REGEN-COV was granted Emergency Use Authorization (EUA) by the U.S. to linearity, accuracy, precision, selectivity, specificity, and analyte stability before and after enzymatic digestion. The designed LC-MRM-MS assay has a dynamic range from 10 to 2000 g/mL antibody drug in the human serum matrix, which was able to cover the serum drug concentration from day 0 to day 28 after drug administration in two-dose groups for the clinical PK study of REGEN-COV. The concentrations of REGEN-COV in the two-dose groups measured by the LC-MRM-MS assay were comparable to the concentrations measured by a fully validated electrochemiluminescence (ECL) immunoassay. Introduction REGEN-COV (REGN10933 + REGN10987, also referred to as casirivimab and imdevimab, respectively) is an investigational antibody cocktail therapy developed by Regeneron Pharmaceuticals, Inc. for the treatment of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).1?3 The antibody cocktail includes two humanized IgG1 monoclonal antibodies (REGN10933 and REGN10987), which are designed to target nonoverlapping epitopes around the SARS-CoV-2 spike protein, thereby blocking the interaction of SARS-CoV-2 virus with human ACE21 and preventing viral escape due to quick genetic mutation of the Levomefolate Calcium virus.4 A recent clinical study has shown that REGEN-COV therapy can reduce viral weight and improve symptoms for nonhospitalized COVID-19 patients, especially those who were seronegative or had high viral loads at baseline.3 Based on the promising results from the clinical investigation, REGEN-COV was granted Emergency Use Authorization (EUA) by the U.S. Food Levomefolate Calcium and Drug Administration (FDA) in November 2020 for the treatment Levomefolate Calcium of recently diagnosed, mild-to-moderate COVID-19 in adults and pediatric patients at Levomefolate Calcium least 12 years of age and weighing at least 40 kg and are at high risk for progressing to severe COVID-19 and/or hospitalization. Due to the urgent need for an effective therapy to treat COVID-19, the timelines for drug discovery and preclinical validation processes of REGEN-COV were highly compressed after the outbreak of the computer virus was designated as a global pandemic. Within 2 months of lead candidate selection for potent neutralizing antibodies against SARS-CoV-2, several clinical trials of REGEN-COV were initiated in hospitalized and ambulatory patients. As part of the clinical study, the determination of circulating drug concentrations in patients is critical for pharmacokinetic (PK) characterization of protein therapeutic and drug dose optimization. To meet this need and manage the accelerated development for any COVID-19 therapy, we developed and qualified a fit-for-purpose liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay for the REGEN-COV PK study in 1 montha much shorter timeframe than that required for the development of a conventional ligand-binding assay. Unlike the ligand-binding assay, the LC-MRM-MS assay does not require highly specific affinity capture and detection reagents for antibody therapeutics, which typically take several months to develop and produce. In addition, the LC-MRM-MS assay also provides a wide dynamic range, good accuracy and precision, excellent selectivity and specificity for the quantification of protein-based biopharmaceuticals in serum matrix.5 Recently, LC-MRM-MS has become a more frequently adopted bioanalytical strategy for both preclinical6?8 and clinical9?11 sample analysis due to continuous improvement in the performance of LC-MS instrumentation. The quantification of total antibody drug concentration, including free and bound antibodies, in human serum samples by LC-MRM-MS assay is based on the measurement of ion intensities of the surrogate peptides derived from the variable complementarity-determining regions (CDRs) Levomefolate Calcium of the antibody drugs.12 To course of action patient serum samples, typically, a few microliters of serum sample was reduced, alkylated, and then underwent protease digestion. Stable heavy isotope-labeled proteins or surrogate peptides are usually used as internal requirements (ISs) to normalize the signal variation from sample processing and instrument performance fluctuation. The sensitivity, selectivity, and specificity of the assay are reliant on the unique CDR peptides that have been selected for quantification. For the REGEN-COV antibody cocktail, the LC-MRM-MS can be readily multiplexed to measure multiple drug analytes simultaneously. Despite limited throughput due to the chromatographic separation, the developed LC-MRM-MS method met the required dynamic range, sensitivity, selectivity, stability, and specificity for the early measurement of drug concentrations of REGEN-COV in a limited number of serum samples in the clinical trials. The method.