International validation of pyrogen tests based on cryopreserved human primary blood cells.

Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1beta; IL-6) upon contact with pyrogens. Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached >90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.

International validation of novel pyrogen tests based on human monocytoid cells.

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.

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.

[Characteristics of Toxoplasma-gondii-tachyzoites from different culture systems]

Toxoplasma gondii is an intracellular protozoan parasite of worldwide distribution. Parasites that habour a complete antigenic profile, that is necessary for the serological diagnosis of human Toxoplasma infections, are provided by in vivo culture methods only. It seems that the host immune pressure is responsible for the expression of a total antigen pattern. Thus, in most laboratories the asexual proliferative stage of the parasite, the tachyzoite, is maintained by successive intraperitoneal passages in highly susceptible animals such as mice. We would like to develop an in vitro method to provide sufficient amounts of high quality parasite antigen suitable for diagnosis of Toxoplasmosis. Using the RAPD-PCR (random amplified polymorphic DNA-PCR) technique, we were able to show that during different culture conditions (in vivo and in vitro culture) the parasites undergo no clonal selection. That means the entire parasite population changes the protein expression pattern due to the in vitro culture conditions. Based on the result described previously the work could be continued as follows. One strategy could be to simulate the host immune pressure during the in vitro cultivation of the tachyzoites. A more convinced approach may be the production of recombinant parasite antigens useful for diagnosis of a Toxoplasma infection in human adults and newborns.