RESUMO
A solution-phase electrochemiluminescence assay (ECLA) was developed to quantify ranibizumab in serum from patients treated with this biotherapeutic for neovascular age-related macular degeneration. Ranibizumab, a recombinant humanized Fab ("fragment, antigen binding"), binds with high affinity and specificity to vascular endothelial growth factor A (VEGF-A), inhibiting its activity. Fab molecules contain the amino acid sequence that binds antigen and are composed of one constant and one variable domain from each heavy and light chain of the antibody. High assay sensitivity was required to enable pharmacokinetic (PK) evaluation of ranibizumab-dosed patients in clinical trials. Our assay's lower limit of quantitation is 300 pg/mL ranibizumab in neat serum, achieving a 67-fold improvement in sensitivity relative to a conventional ELISA-based PK method. In this assay, ruthenium-labeled affinity-purified rabbit anti-ranibizumab antibodies and biotinylated rhVEGF are added to serum samples. During overnight incubation, these two labeled molecules bind to ranibizumab, and the resulting immune complex is then captured by streptavidin-coated paramagnetic beads and analyzed for electrochemiluminescence. The ranibizumab PK ECLA has a reporting range of 300-24,000 pg/mL, based on accuracy and precision parameters. It showed high precision for both intra- and inter-assay analyses. Recovery of ranibizumab from 10 individual donors averaged between 100% and 119% of nominal concentration. There was no cross-reactivity observed in the assay to other recombinant humanized antibodies (whole molecules or monoclonal antibody fragments) or human IgG. To our knowledge, this report represents the first description of development and validation of an ECLA-based PK assay for a recombinant humanized Fab therapeutic agent.