Translational Medicine

Validation of a Gyrolab™ Assay for Quantification of Rituximab in Human Serum

The gold standard for PK analysis of macromolecules is the ELISA (enzyme-linked immunosorbent assay). However, ELISAs are labor intensive, expend large amounts of precious reagent, and often have a narrow range of quantification, which makes the necessity for re-assay more likely, especially in the face of high inter-individual PK variability. More efficient, newer methods with wider ranges and quicker turnaround would be useful for large-scale late-phase clinical trials.

The Gyrolab™ is a rapid, fully automated nanoscale immunoassay platform with good reproducibility and data quality. However, few Gyrolab™ assays have been fully validated. In this study, QPS developed and validated a Gyrolab™ assay for Rituximab levels in human serum. Rituximab is a monoclonal antibody that binds either normal or malignant B-lymphocytes, used in the treatment of lymphoid cell tumors and rheumatoid arthritis. The pharmacokinetics of Rituximab are highly variable from person to person, so studies of Rituximab would benefit from an assay with a wider range of quantification than ELISA.

The validation assay followed a sandwich immunoassay format: rat anti-idiotypic Rituximab antibody was biotinylated, then reacted with the streptavidin-coated beads in the Gyrolab™ Bioaffy compact disk to form the capture antibody. The detection antibody consisted of goat anti-human IgG labeled with Alexa Fluor™.

Calibration standards, validation samples, and quality controls (QC) in concentrations ranging from 90-60,000 ng/ml were prepared by spiking Rituximab in pooled human serum. Sera from healthy volunteers and solid tumor patients were tested.

The authors referred to industry immunoassay guidelines to set the criteria for run and method acceptance. After assessing such parameters as method precision and accuracy, dilutional linearity, selectivity, and analyte stability, the authors concluded that the Gyrolab™ assay is, indeed, valid for detecting Rituximab levels in human sera, with selectivity comparable to ELISA and a tenfold wider range, and represents a high quality, high-throughput methodology for late phase clinical trials with large sample numbers.

Validation of a Gyrolab™ Assay for Quantification of Rituximab in Human Serum

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