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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.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) pointed out the need to involve authorities throughout the process of validation and legal acceptance of alternatives to animal experiments. The Paul-Ehrlich-Institute (PEI), Federal Agency for Sera and Vaccines, is the national competent authority in Germany which is responsible for the quality and safety of biologicals including blood and cell-based products. This paper is intended to contribute to the discussion concerning the use of alternative methods in safety testing of medicinal products and considers the scientific work of the PEI in this field. From a regulator's perspective, adequate demonstration of safety and quality of medicinal products are of major interest. Additionally, the availability of the products to the patient has to be taken into consideration. It has to be carefully explored whether the respective in vitro method for demonstration of non-clinical safety as part of the non-clinical development programme is able to guarantee safety level comparable to the corresponding experiment in animals. The topics cited above shall be discussed in this paper using the example of the Alternative Pyrogen Test or also called Monocyte Activation Test. The Alternative Pyrogen Test could serve as paradigm to exemplify how an alternative test can provide at least a comparable level of safety estimation in comparison with a conventional animal test. Furthermore, this alternative test creates additional information which cannot be obtained from the animal experiment, and might also open further scientific insight into the mechanisms of pyrogenicity and acute pro-inflammatory reactions in patients. This test method allows the definition of pyrogen limits for medicinal products. Due to its use of relevant cell systems this in vitro test might contribute significantly to safety assessments of advanced medicinal products during the pre-clinical phase.

A comparative study of rabbit pyrogen test and human whole blood assay was performed on released preparations of human serum albumin. In addition, the samples were spiked with 5 IU/ml (in whole blood 0.5 IU/ml too) and 10 IU endotoxin/ml. The unspiked samples were negative in both assays. The human whole blood test resulted in the same level of security for the products as the rabbit pyrogen test did. Both, the borderline 5 IU/kg and the 10 IU/kg-Spike partially lead to results of the rabbit test which would cause further testing with additional animals. In contrast, the human whole blood assay resulted in a 100% detection for the 5 IU/ml and 10 IU/ml-Spike. We designed a study protocol for a minimised number of test animals and were able to show the general usefulness of the human whole blood assay.