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1.
《MABS-AUSTIN》2013,5(4):1103-1113
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG2 antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use. 相似文献
2.
Quantitative analysis of organophosphorus pesticides in freshwater using an optimized firefly luciferase‐based coupled bioluminescent assay
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In this paper, a coupled bioluminescent assay, relying on the coupling of the enzymes acetylcholinesterase, S‐acetyl‐coenzyme A synthetase and firefly luciferase, for the detection and quantitation of organophosphorus pesticides, is presented. Using malathion as a model organophosphorus pesticide, the assay was optimized through statistical experimental design methodology, namely Plackett–Burman and central composite designs. The optimized method requires only 20 μL of sample. The linear range for the assay was 2.5–15 μM of malathion, with limits of detection and quantitation of 1.5 and 5.0 μM, respectively. This simple, fast and robust method allows samples to be analyzed at room temperature and without any pretreatment. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
3.
Alexander Colbert Amber Umble-Romero Samantha Prokop Ren Xu John P Gibbs Susan Pederson 《MABS-AUSTIN》2014,6(4):1103-1113
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG2 antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use. 相似文献
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Nanyan Rena Zhang Nathan G. Hatcher Kim Ekroos Komal Kedia Monika Kandebo Jacob N. Marcus Sean M. Smith Kevin P. Bateman Daniel S. Spellman 《Journal of lipid research》2022,63(6):100218
A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery. 相似文献
7.
In recent years, the applicability of using LC-MS/MS as a complementary technique to traditional ligand binding assays in the absolute quantitation of therapeutic proteins in biologic matrix has been demonstrated. Protein quantitation workflow via LC-MS/MS is primarily based on a enzymatic digestion model and recent works seek to improve selectivity and sensitivity. This review focuses on recent innovations in this field and discusses the following in detail: the applicability of two-dimensional liquid chromatography and its use to improve sensitivity and alleviate matrix ion suppression; the use of derivatization agents after digestion to improve extraction and MS ionization efficiency; techniques to reduce excess protein background and their positive effects on sensitivity, selectivity, and extraction consistency; the application of immunoaffinity extraction of proteins to enrich the analyte(s) of interest while improving selectivity and sensitivity. 相似文献
8.
《MABS-AUSTIN》2013,5(5):1178-1189
The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development. 相似文献
9.
Said A. Hassan Aya H. Helmy Nadia F. Youssef Soheir A. Weshahy Badr A. El-zeany 《Luminescence》2023,38(6):729-735
Antiepileptic drugs are among the most common medications that require therapeutic drug monitoring (TDM). Indeed, TDM provides a realistic approach to adjust drug doses for epilepsy based on plasma concentrations to optimize its clinical outcome. The most common technique for TDM is high-performance liquid chromatography, which has a very low green profile among analytical techniques. Perampanel (PER) is an inherently fluorescent compound that its fluorophore readily allows sensitive and quantitative measurements. This paper describes the development and validation of a sensitive, specific, and eco-friendly spectrofluorimetric method for the determination of PER. Experimental parameters affecting fluorescence intensity of the compound, including solvent dilution, temperature, and excitation wavelength, were studied and optimized. The developed spectrofluorimetric method was established in acetonitrile at λex = 295 nm and λem = 431 nm over a concentration range of 5–60 ng/ml. The adopted method was applied for the determination of PER in human plasma; it was effective in the range of 15–50 ng/ml. The proposed method was found to be sensitive and specific for PER and can be applied successfully in TDM of PER and in quality control laboratories. 相似文献
10.
Alex Colbert Amber Umble-Romero Samantha Prokop Vincent Fung-Sing Chow Teresa Wong Danielle DeSimone Lei Zhou Susan Pederson 《MABS-AUSTIN》2014,6(5):1178-1189
The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development. 相似文献