Development and validation of the IFN-γ FlowCytoScreen for cytokine-based potency assessment of advanced therapy medicinal products.

Abstract

Robust quality control methods are required to ensure the safety, efficacy and consistency of advanced therapy medicinal products. Potency assays play a critical role in evaluating their biological activity. While ELISpot remains the reference method for single-cell cytokine analysis, it has several limitations such as a lack of standardisation and limited capacity for multiparametric characterisation. This study aimed to develop and validate a flow cytometry-based potency assay for the assessment of single-cell IFN-γ secretion. The commercially available Miltenyi Biotec IFN-γ Secretion Assay - Detection Kit, based on flow cytometry, was modified to assess the potency of dendritic cell-based vaccines. To ensure its reliability, our assay was rigorously evaluated for specificity, linearity, accuracy, robustness and precision. Comparative analysis with the ELISpot assay was also performed to assess agreement between the two methods. Method development resulted in reduced sample requirements and optimisation of cytokine secretion conditions to generate a response to antigen-presenting cell stimulus. Validation showed that the specificity, linearity, accuracy, robustness and precision consistently met the acceptance criteria. Comparative analysis with ELISpot demonstrated high correlation and agreement, with no evidence of systematic or proportional error. The developed assay provides a reliable, scalable and efficient flow cytometry-based approach for cytokine secretion analysis and represents an alternative method to ELISpot. Its advantages in sensitivity, efficiency and cost effectiveness make it a valuable tool for advanced therapy quality control and immune monitoring in clinical research. The flow cytometry further enhances its potential by enabling multiparametric immune profiling, supporting comprehensive characterisation of cellular products in both preclinical and clinical applications.