Validation of an ELISA for the determination of rituximab pharmacokinetics in clinical trials subjects

Abstract

Rituximab is a chimeric anti-CD20 monoclonal antibody that has revolutionised the treatment of many B-cell malignancies, and is now increasingly being used in non-malignant conditions such as auto-immune disorders. Serum rituximab levels are highly variable in patients receiving similar 'standard' approved doses. Little is known regarding the factors that affect serum rituximab concentration and that in turn may influence clinical outcome. In order to provide a tool that may ultimately enable patient specific dosing of rituximab therapy, we have validated a reliable, robust ELISA for the quantitation of serum rituximab levels to provide accurate pharmacokinetic (PK) data that will guide the optimisation of rituximab dosing regimes. Extensive validation of the assay was performed in order to utilise the assay for clinical applications. The within and between day plate coating reproducibility was tested and proved a robust starting platform for the assay. The within day precision for the assay was determined using spiked serum samples and was shown to have a coefficient of variation (CV) of <10% with an accuracy between 91 and 125%. The between day precision (CV) was <25% with an accuracy between 95 and 109%. Dilution linearity and parallelism were demonstrated. Spike recovery for all concentrations and donors was shown to be within +/-15% on average, with a CV below 10%. This assay is highly accurate and reproducible in determining the levels of rituximab in spiked serum samples. It meets stringent acceptance criteria, is fit for purpose, and is currently being applied to several clinical trials incorporating rituximab in the treatment of lymphoma. This assay represents a useful tool for clinical application of this widely used therapeutic.