As the number of therapeutic protein products is growing rapidly, there is a strong need for the development of bioanalytical methods that are easy to perform, specific, sensitive, robust, and affordable. Methods for immunogenicity evaluation of therapeutic proteins take an important place in this field of bioanalytics. The aim of the study was to develop a method for immunogenicity testing of the novel RPH-104 drug using the Affinity Capture Elution (ACE) ELISA technique. RPH-104 is a promising Interleukin-1 (IL-1) inhibitor that is currently undergoing a series of clinical studies, including those on socially significant and orphan diseases. The developed method was validated for assay cut-point, sensitivity, selectivity, drug tolerance, hook effect, specificity, precision, and stability. Method sensitivity was established at 114.9 ng/mL, while low and high positive controls were equal to anti-RPH-104 antibody concentrations of 155 ng/mL and 2500 ng/mL, respectively. Method specificity was confirmed in the presence of the interfering compounds, namely IL-1α, IL-1β, and IL1-Ra. The developed and validated ELISA method was successfully applied to subject samples.
Keywords: ACE; ADA; ELISA; RPH-104; bioanalytical method; immunogenicity; interleukin-1; validation.