Validation of a test system for the assessment of in vitro cytokine release with human peripheral blood mononuclear cells

CBF 2.0 2025 -- Cytokine release syndrome (CRS) is a potentially life-threatening systemic inflammatory response triggered by the rapid release of cytokines, often observed following immunotherapies such as monoclonal antibodies, bispecific T-cell engagers and CAR-T cell therapies. Accurate prediction and monitoring of CRS risk are critical for ensuring patient safety during clinical development. The cytokine release assay (CRA) is a pivotal in vitro method used to evaluate the potential of therapeutic candidates to induce cytokine release from immune effector cells. By measuring key cytokines, CRA provides essential insights into the immuno-stimulatory profile of biologics. Here we present a validation of a robust CRA method with both a solid and liquid phase designed to measure a wide range of cytokines/chemokines to support preclinical safety assessment. The objective is to validate a method for the measurement of cytokine release in human peripheral blood mononuclear cells (PBMCs) in formats of wet coat (solid phase) and high-density cell pre-culture (liquid phase), and to determine if the test system consisting of two positive controls causes the release of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-γ, TNF-α and MCP-1. The validation process encompasses kit assessment, sensitivity, precision and reproducibility. Technical challenges that were evaluated during validation are described herein.