Binding and cleavage (BINACLE) assay for the functional in vitro detection of tetanus toxin: applicability as alternative method for the safety testing of tetanus toxoids during vaccine production

Abstract

Tetanus toxoids (i.e. chemically inactivated preparations of tetanus neurotoxin) are used for the production of tetanus vaccines. In order to exclude the risk of residual toxicity or of a "reversion to toxicity", each batch of tetanus toxoid is subject to strict safety testing. Up to now, these prescribed safety tests have to be performed as in vivo toxicity tests in guinea pigs. However, as animal tests are generally slow, costly and ethically disputable, a replacement by an in vitro method would be desirable. A suitable alternative method would have to be able to sensitively detect already low concentrations of active tetanus neurotoxin in matrices containing large amounts of inactivated toxoid molecules. We have developed a method which detects active tetanus neurotoxin molecules based on their specific receptor-binding capacity as well as their proteolytic activity. By taking into account two relevant functional characteristics, this combined "BINding And CLEavage" (BINACLE) assay more reliably discriminates between toxic and detoxified molecules than other in vitro assays which solely rely on one single toxin function (e.g. endopeptidase assays). Data from an in-house validation show that the BINACLE assay is able to detect active tetanus neurotoxin with a detection limit comparable to the in vivo test. The sensitive detection of active toxin which has been spiked into toxoid samples from different manufacturers could also be demonstrated. Specificity and precision of the method have been shown to be satisfactory. The presented data indicate that for toxoid batches from some of the most relevant European vaccine manufacturers, the BINACLE assay may represent a potential alternative to the prescribed animal safety tests. In addition, this novel method may also provide a convenient tool for monitoring batch-to-batch consistency during toxoid production.

Keywords: 1,4-piperazinediethanesulfonic acid; BINACLE assay; BSA; CV; DL; EP; European Pharmacopoeia; Ganglioside GT1b; H-chain; In vitro toxicity assay; L-chain; PBS; PIPES; Synaptobrevin; TCEP; TMAO; TeNT; Tetanus neurotoxin; Toxoids; Tris(2-carboxyethyl)-phosphine hydrochloride; binding and cleavage assay; bovine serum albumin; coefficient of variation; detection limit; heavy chain; light chain; phosphate buffered saline; rSyb2; recombinant synaptobrevin-2; tetanus neurotoxin; trimethylamine-N-oxide.