Development of an in vitro Biochemical Assay System for the Measurement of Residual Toxin Activities in Pertussis Toxin Containing Vaccines

Pertussis (whooping cough) vaccines play an essential role in immunisation programmes worldwide and the histamine sensitisation test (HIST) is currently the official batch-release test for acellular pertussis containing combination vaccines. HIST, being a murine animal test, has large assay variability problems and ethical issues. Therefore, in response to the 3Rs, a biochemical assay system has been developed based on the pertussis toxin (PTx)’s two functional domains: an enzyme active A-protomer and a carbohydrate binding B-oligomer. The clinical relevance of these two biochemical tests in relation to the PTx toxic effects has been demonstrated. In order to assess the practicality of this assay system, an international collaborative study involving 9 countries on method transferability had also been performed and showed that this assay system could be adopted readily in control laboratories and vaccine manufacturing facilities. In this update, we provide further optimised assay conditions for measuring both the residual PTx ADP-ribosyltransferase and carbohydrate binding activities in specific vaccines. In general, appropriate dilution of vaccines would help to increase test sensitivity. Spiking experiments showed carbohydrate binding assay could detect low levels of PTx similar to HIST. Although positive trend with increasing spikes could also be demonstrated in ADP-ribosyltransferase activity assay for some vaccines, the resultant values in a number of spiked vaccines were not statistically significant and this is due to high baseline activity of the unspiked vaccines. Since all the reagents of this biochemical assay system are available worldwide, readily controlled and with good test consistency, both assays could be used for monitoring product consistency. The carbohydrate binding assay could even be considered as a potential alternative to HIST for batch-release of acellular pertussis vaccines. Protocols for this in vitro assay system are supplied as Supplement Documents 1 & 2.