Bordetella Pertussis Toxin does not induce the release of pro-inflammatory cytokines in human whole blood.

Pertussis Toxin (PTx) is one of the most important virulence factors of Bordetella pertussis, the cause of whooping cough. Therefore, the inactivated toxin is an obligatory constituent of acellular pertussis vaccines. It is described in the literature that both native PTx and recombinant Pertussis Toxin (PTg) activate human monocytes whereas others report an inhibition of mammalian monocytes during pertussis infection. B. pertussis, as a Gram-negative bacterium, harbours naturally lipopolysaccharide (LPS, also known as endotoxin), one of the strongest stimulators of monocytes. The latter is triggered via the interaction of endotoxin with inter alia the surface receptor CD14. Consequently, it is necessary to consider a potential contamination of Pertussis Toxin preparations with LPS. First, we determined the LPS content in different preparations of PTx and PTg. All preparations examined were contaminated with LPS; therefore, possible PTx- and PTg-driven monocyte activation independently of LPS was investigated. To meet these aims, we examined monocyte response to PTx and PTg while blocking the LPS receptor CD14 with a specific monoclonal antibody (anti-CD14 mAb). In addition, all toxin preparations examined underwent an LPS depletion. Our results show that it is contaminating LPS, not Pertussis Toxin, which activates human monocytes. Blocking the CD14 receptor prevents Pertussis Toxin-mediated induction of pro-inflammatory cytokines in human monocytes. The depletion of LPS from Pertussis Toxin leads to the same effect. Additionally, the PTx toxicity after LPS depletion procedure was confirmed by animal tests. In contrast, the original Pertussis Toxin preparations not treated as mentioned above generate strong monocyte activation. The results in this publication allow the conclusion that purified Pertussis Toxin preparations do not induce the release of pro-inflammatory cytokines in human whole blood.

Microbial safety of cell based medicinal products--what can we learn from cellular blood components?

Today, sterility of established parenteral drugs including biologicals, such as plasma derived products, is practically guaranteed. Bacterially contaminated products are extremely rare exceptions owing to the efficiency of the manufacturing processes in the pharmaceutical industry. In contrast, the manufacturing processes of cell based medicinal products or tissue preparations show much less defined conditions. The sterility of source materials cannot be guaranteed in many cases. As a rule, these source materials cannot be sterilised, as it holds true for the final products. Furthermore, the established methods for sterility testing are not applicable for cell preparations. Sterility of a restricted sample does not guarantee sterility of the whole preparation. Thus, small amounts of residual bacteria in the product can be overlooked and can grow up to enormous numbers during storage and shipping of cell based medicinal products. Considering these problems, there are some parallels in the warranty of microbial safety of cellular blood components. Therefore, the experiences collected in transfusion medicine in the past decade can be successfully used in the production of cell based medicinal products. Comparable to the situation regarding cellular blood components, there is a need for new principles in rapid bacteria detection.

Safety testing of cell-based medicinal products: opportunities for the monocyte activation test for pyrogens.

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.

[Alternatives to animal experimentation. The sophisticated process of validation]

The monographs of the European Pharmacopoeia (Ph.-Eur.) are legally binding. Namely in immunobiologicals there is still need to perform animal trials in testing for safety and potency. Only an alternative method which is introduced into the Ph.-Eur. is able to substantially reduce the number of animals. To introduce a new method into the Ph.-Eur., mainly to substitute the assay but also as part of the test- or in process-section of the monographs, the requirements of a formal follow up have to be met. To understand these mechanisms, some of the underlying principles and the way a monograph is established are explained. To meet the requirements of a validated alternative method the steps from the first idea to the request for revision are recapitulated. The development of such a method should be performed in close co-operation with the respective group of experts and the biological standardisation group of the Ph.-Eur. Some ideas for acceleration of the implementation are given.

[Is the abnormal toxicity test still relevant for the safety of vaccines, sera and immunoglobulins?]

The German Pharmacopoeia (DAB) requires the abnormal toxicity test (ATT) using mice and guinea pigs as a non-specific safety test for vaccines, sera and immuno-globulines. The purpose of this project was to investigate the relevance of ATT after the introduction of GMP- and GLP-principles in the manufacturing of biological products. A great variability in the test performance became evident for the different test laboratories, involving the animal number as well as the vaccine dosage administration and test duration. The retrospective analysis of ATT results reveals reasons for the incompatibility of particular preparation groups, vaccine components or additives with the animal species used. There were highly significant differences between the manufacturers and the PEI regarding the frequency of deviating test results for identical test batches. Positive ATT"s never resulted from the insufficient quality of a batch. On the other hand vaccines causing adverse reactions in the target species were not identified by the ATT. For these reasons the abnormal toxicity test is unsuitable to detect harmful batches. The results of the analysis of data show that finally the ATT has always been passed, i.e. there was no retention by the vaccine manufacturers and no refusal by the controlling authorities due to the ATT-results. Considering the present animal model and the questionable transferability of the test results to the target species only a poor reliability is evident. Taking into account aspects of drug safety and animal welfare it is recommended to the DAB to omit the ATT.