Detection of Cell Impurities and NF-κB activation Using SEAP Reporter Cell Lines in Vaccines.

INTRODUCTION During the 2010 influenza season, the Therapeutic Goods Administration (TGA) reviewed adverse event (AE) signals of febrile convulsions in children following the administration of the seasonal Fluvax® trivalent vaccine. These AEs were attributed to 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. Agonists for each cell line demonstrated linearity within the working range, met precision criteria for repeatability and precision. The assay tested viral protein, messenger RNA (mRNA), and bacterial protein vaccines, 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 This assay 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.