Development and validation of a novel semi-homogenous clinical assay for quantitation of Ranibizumab in human serum.

Ranibizumab (Lucentis®), a humanized antigen-binding fragment (Fab) monoclonal antibody that blocks VEGF-A activity, is currently approved for the treatment of several retinal degenerative diseases. The assessment of drug pharmacokinetics (PK) is essential for evaluating exposure as it relates to drug safety and efficacy. For drugs administered intravitreally, systemic drug levels during the course of clinical studies are typically 100 to 1000-fold lower than those of similar therapeutics dosed intravenously, posing a significant bioanalytical challenge for PK measurements. Thus, the development of a highly-sensitive assay for measuring pg/mL levels of ranibizumab in patients' sera after intravitreal administration was needed to support clinical studies. In this report, we describe the development of a novel method that utilizes a high-affinity murine monoclonal anti-ranibizumab-VEGF-complexes antibody (MARA) reagent to measure ranibizumab in human serum. The assay format utilizes a semi-homogeneous solution phase step using a monoclonal antibody (the MARA) that binds specifically to the ranibizumab-VEGF complex, but not to either alone. This unique reagent exhibited low non-specific binding and high selectivity, increasing signal-to-noise readouts and maximizing assay sensitivity. The resulting MARA enzyme-linked immunosorbent assay (ELISA) has a lower limit of quantification of 15 pg/mL in human serum. In the assay, serum samples are incubated overnight with a mixture containing biotinylated-VEGF and MARA, which form a three-molecule complex with ranibizumab in the sample. These complexes are then captured onto streptavidin-coated wells, followed by enzymatic detection using a horseradish peroxidase-labeled-anti-murine antibody reagent and a colorimetric reaction. The assay conditions were optimized to allow for quantitative detection of "total" ranibizumab levels in serum. The assay was fully validated, establishing its high tolerance to sample matrix, as well as its suitable specificity, accuracy, dilution linearity, as well as intra- and inter-assay precision. The MARA ELISA's novel and unique approach has resulted in a considerably more sensitive ranibizumab PK assay compared to earlier versions of this assay. The MARA ELISA has been used for PK measurements in multiple ranibizumab studies, supporting this drug's life-cycle management and related preclinical and clinical-development studies.

Development of a novel homogenous electrochemiluminescence assay for quantitation of ranibizumab in human serum.

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.

Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration.

PURPOSE: Ranibizumab (rhuFab V2; Lucentis, Genentech, South San Francisco, CA) is a humanized monoclonal antibody fragment designed to bind all forms of VEGF, thereby blocking vessel permeability and angiogenesis in neovascular age-related macular degeneration. This study evaluated the pharmacokinetic (PK) and serum bioavailability of ranibizumab after a single intravitreal (ITV) or intravenous (IV) dose in cynomolgus monkeys. METHODS: Monkeys received ranibizumab as either a bilateral ITV dose (500 or 2000 microg/eye; n = 6/group) or a single IV dose (1000 or 4000 microg/animal; n = 4/group). After ITV administration, ranibizumab concentrations were measured in several ocular compartments and in serum for 10 days and, after IV administration, for 48 hours. Pharmacokinetic parameters were estimated by compartmental and noncompartmental methods. RESULTS: Ranibizumab cleared in parallel from all ocular compartments, with a terminal half-life of approximately 3 days. It distributed rapidly to the retina (6-24 hours), and concentrations were approximately one third that in the vitreous. After ITV injection, bioavailability (F) was 50% to 60%. Serum concentrations were very low, reflecting wider distribution and faster clearance when ranibizumab reached the serum. After IV administration, the terminal half-life was approximately 0.5 day. CONCLUSIONS: This study demonstrates that ranibizumab has a PK profile that is favorable for its clinical use in treating neovascular AMD by monthly ITV injection.