Detection of Cell Impurities and Nf-Κb Activation Using Seap Reporter Cell Lines in Vaccines

Abstract

Introduction: During the 2010 influenza season, the Therapeutic Goods Administration (TGA) reviewed post-market adverse event (AE) signals of febrile convulsions in children under five years old following the administration of the seasonal Fluvax® trivalent vaccine. These AEs were attributed to elevated cytokine/chemokine secretion via nuclear factor kappa-beta (NF-κB) activation. In response, the TGA developed a method to detect NF-κB activation by vaccine samples.Methods: Complementary Toll-Like Receptor (TLR)-expressing reporter cell lines THP1-Blue and Ramos-Blue enabled detection of TLR1-9 activation using the QUANTI-Blue™ system following incubation with a panel of TLR agonists, tumour necrosis factor-alpha (TNF-α), or vaccine sample.Results: Using TNF-α as a positive control and specific Toll-like receptor (TLR) agonists, specificity was confirmed for each cell line based on extracellular (THP1-Blue) or endosomal (Ramos-Blue) TLR expression. Selected agonists for each cell line demonstrated linearity within the working range and met precision criteria for repeatability and intermediate precision. The assay, tested with various vaccine types (viral protein, mRNA, and bacterial protein), met accuracy parameter of 80-120% recovery of spiked controls.Discussion: We established and validated a robust, sensitive and reliable analytical approach for detecting possible NF-κB activation by bioactive components in vaccines.Interpretation: Our validation method is applicable to influenza, SARS-CoV-2 and bacterial vaccines demonstrating excellent specificity, repeatability, and accuracy per ICH guidelines. Our findings indicate that this technique, a valuable addition to the regulatory arsenal, not only provides an effective means to assess vaccine quality in relation to impurities but also suggests potential broader applications across various vaccine types. This enables manufacturers and regulators to meet the growing demand for reliable impurity screening assays that support the characterization of vaccines activating the NF-κB pathway. Future studies must further characterise NF-κB activation by individual TLRs across a broader range of vaccines.

Note:
Funding declaration: No external funding was provided for this study.

Conflict of Interests: All authors of this article were employees of the Therapeutic Goods Administration within the Department of Health and Aged Care at the time of all data collection for the article.

Keywords: NF-κB, Vaccine, Cell Impurities, Method Development