Optimization of the monocyte-activation-test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine.

Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: 1) the Rabbit Pyrogen Test (RPT), 2) the Bacterial Endotoxin Test, 3) the Recombinant Factor C test and the Monocyte Activation Test (MAT). Here, we explored the possibility to replace RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cellular source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the set-up of a very sensitive assay (limit of detection, LOD = 0.04 EU/ml; Sensitivity = 0.1 EU/ml). Both quantitative Method A and semi-quantitative Method B were used for data analysis. Our studies revealed that for vaccine without intrinsic pyrogenicity, as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD in order to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current MAT chapter 2.6.30 and propose a re-evaluation of the validity criteria of the Methods A and B, in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.