A risk assessment on extraneous virus contamination in a viral vaccine production environment justifies waiving of in vivo testing.

For many years in vivo assays have been a corner stone in safety testing of vaccines for human use. However, there is now an increasing regulatory focus on replacement, reduction and refinement of methods involving animal use. Accordingly, European Pharmacopoeia (Ph.Eur.) monographs and chapters are currently being revised to reduce or discontinue the use of animals in safety and other testing, when such in vivo tests are not absolutely necessary to facilitate risk mitigation. In the current study, a risk assessment of extraneous agents in viral vaccine production has been carried out and it is concluded that only the handling procedures carried out by the technical personnel pose a risk for extraneous viral contamination. A list of named, potentially virulent contaminating viruses, which may have been introduced by these procedures, has been generated. Each of the viruses on this list has been evaluated for possible persistence during the production processes, and it has for all of these been concluded that, if at all present, they only present a negligible risk of introducing extraneous agents in the final product. The overall conclusion of the risk assessment of our vaccine production process is that it justifies the discontinuation of the current in vivo testing, and furthermore demonstrates that there is no need to substitute these in vivo assays with novel in vitro methods.

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines.

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.

Development and validation of an ATP method for rapid estimation of viable units in lyophilised BCG Danish 1331 vaccine.

An assay for quantifying viability in BCG vaccine by determining intracellular ATP content was developed and validated. ATP content was determined by measuring bioluminescence in the presence of luciferin/luciferase. During development and validation the ATP method was compared to the conventional viable count method. A key step to obtain correlation between ATP content and CFU was found to be a period of pre-incubation in a growth medium before ATP determination. During the validation, the robustness, linearity, accuracy, precision, and range were studied. The method validation study showed that the method applied was robust and applicable to determine ATP content in lyophilised BCG for estimating viability in the BCG samples. By comparison with a conventional viable count method, a high correlation between ATP content and the viable count was found; this relationship can be applied in routine quality control to estimate viable count from the ATP content determined in a sample.