Validation of a gyrolab™ assay for quantification of rituximab in human serum

Gyrolab™ technology presents a technology breakthrough for large molecule bioanalysis to support biologic drug development. The advantages of this innovative platform include fully automated nanoscale immunoassay capability, better assay reproducibility and data quality, small reagent and sample volumes, and rapid assay development and validation as a result of reduced run time. Although Gyrolab has been increasingly used in method development in discovery environment, few fully validated Gyrolab assays have been reported. Here we report a method validation of a Gyrolab assay to determine rituximab levels in human serum. Rituximab is captured on a Bioaffy™ CD by a biotinylated rat anti-idiotypic monoclonal antibody against rituximab and detected by an Alexa Fluor®-labeled anti-human IgG antibody. Assay conditions were optimized to give required sensitivity and dynamic range. The assay validation was conducted according to the current industry standards for GLP-regulated immunoassays. The intrabatch precision and accuracy for the assay were determined using spiked human serum samples and shown to have a coefficient of variation (CV) of < 11% with a mean bias < 20%. The interbatch precision (CV) and absolute mean bias were both < 12% with the total error < 25%. Adequate spike recovery was demonstrated in serum samples of healthy individuals and solid tumor patients. The dilutional linearity test showed that the determined concentrations adjusted with various dilution factors had a linear relationship with the expected concentrations and that there was no hook effect. The method has been validated for the quantification of rituximab in human serum from 90 to 60,000 ng/mL with a minimum required dilution of 30. The Gyrolab assay was proved to be accurate, precise and selective, with a comparable sensitivity to the ELISA method, but provided an automated nanoscale assay with a significantly wider assay dynamic range for the determination of rituximab in human serum during pharmacokinetics/toxicokinetics studies.