Process Development for a Veterinary Vaccine Against Heartwater Using Stirred Tanks

Abstract

The work presented herein reports the improvements achieved on endothelial cell culture conditions and the production of Ehrlichia ruminantium (ER) under stirring conditions towards the development of a cost effective process for the widespread application of the inactivated vaccine against Heartwater. The effect of cell origin, inoculum size and microcarrier type upon maximum cell concentration was evaluated. Afterwards, using the optimised parameters for cell growth, ER production in stirred tanks was validated for two bacterial strains (Gardel and Welgevonden). Critical bioprocess parameters related with the infection strategy such as serum concentration at time of infection, multiplicity of infection, and medium refeeding strategy were analysed. The results obtained indicate that it is possible to produce ER in stirred tank bioreactors and that the production yields can be increased by a factor of 6.5 using a serum-free medium during and after the infection process. To expand the scale-up process and improve ER production yields, ER growth kinetics was characterised showing that this stirring culture system is capable of efficient bacterial amplification with maximum titers going up to 2.2 log10ER. The suitability of this process was validated up to a 2L scale. A downstream processing for the purification of ER is also described, taking into account ER recovery yields and the number of steps. Overall, these results open "new avenues" for the production of other ehrlichial species, with emerging impact in human and animal health, in a fully controlled and scaleable environment.