Fed-batch cultivation of Bordetella pertussis: metabolism and Pertussis Toxin production.

The production of acellular pertussis in comparison with whole cell pertussis vaccines demands 5-25 times the amount of Bordetella pertussis' virulence factors, such as Pertussis Toxin (PT), to produce the same number of vaccine doses. An increase in the volumetric productivity by employing fed-batch rather than the currently used batch cultivations of B. pertussis could reduce the cost price of acellular pertussis vaccines. This study defined the conditions that enable fed-batch cultivations at high specific PT production. A solution containing lactate and glutamate was fed to the cultures at various rates. The feed rate and whether or not the fed substrates were completely consumed, significantly influenced cellular metabolism. If lactate was detectable in the culture broth while glutamate was not, poly-hydroxy-butyrate (PHB) was formed. Any PHB present was metabolized when glutamate became detectable again in the culture liquid. At higher lactate and glutamate concentrations, free fatty acids were produced. Though toxic, free fatty acids were not the reason the cultures stopped growing. By choosing appropriate conditions, a cell density of 6.5 g/L dry weight was reached, i.e. a 7-fold increase compared to batch culture. The metabolic mechanisms behind the formation of PHB and fatty acids are discussed, as well as how to increase the cell density further. The PT production stopped at 12 mg/L, well before growth stopped, indicating that regulatory mechanisms of PT production may be involved.

Effect of relevant culture parameters on Pertussis Toxin expression by Bordetella pertussis.

Whooping cough vaccines are produced using different ranges of cultivation conditions and medium compositions, which are known to influence growth rate, virulence factor production and degradation, as well as the virulence factors' association to the cell. This study quantifies the impact of individual parameters on Pertussis Toxin (PT) production, using an optimized chemically defined medium as starting point, rather than a complex medium. A number of chemicals that are identified affect both growth rate and virulence factor production, which occur at similar levels in various commonly used production media. Also, degradation by proteolytic activity is shown to be an important parameter to monitor, since it significantly affects the PT yield. Low sodium concentrations, i.e. 50-75 mM rather than the conventional 100-140 mM, significantly increase the growth rate of the organism, the final optical density, as well as the association of PT to the cells. The absolute amount of biomass produced measured as dry weight, is similar for all sodium concentrations tested, contrary to earlier work. While it is known that high iron concentrations inhibit virulence factor production, it is shown here that iron-limited growth results in very high specific PT production. This finding may be used to produce a whole-cell vaccine with little biomass per dose, reducing whole-cell vaccine toxicity. The Bordetella pertussis strain 509 used here produces 30% more PT at 34 than at 37 degrees C, a commonly used cultivation temperature. The data in this study show that existing production processes for cellular and acellular vaccines can in principle be optimised considerably by taking simple measures.