Abstract

Negative effects of the tick Boophilus microplus on cattle vaccinated with the natural protein, Bm86, isolated from the tick gut epithelial cells, have been progressively reduced. Gene coding for the protein Bm86 has been cloned and expressed in the yeastPichia pastoris and a technology has been developed to obtain the vaccine Gavac™ for the control of cattle tick. An unexpected misstargeting of the recombinant protein took place during fermentation process and the protein remained held into the cell strongly associated to the insoluble fractions of the yeast. In consequence, stages to isolate the target protein involved cell washing and harvesting, cell disruption, pellet-cell washing, solubilization, and ultrafiltration. Scale up of mechanical cell disruption was successfully achieved, from a laboratory bead mill to a pilot plant mill, keeping constant the total retention time. Modelling of pellet-cell washing allowed finding optimal conditions to remove 88.9 and 63% of contaminant proteins at laboratory and pilot plant scales, respectively. Diafiltration was found to be the most economic operation to remove urea from denatured protein solution. Statistical analysis of 18 production batches showed that rBm86 extraction process is under control. Here we report the scale up criteria applied in recovery steps and results are discussed regarding to each unit operation, as a contribution to the scale-up methodology for downstream processes.

Key words: Scale-up, downstream process, Bm86 protein, Pichia pastoris, vaccine.