Proteon Pharmaceuticals

Proteon Pharmaceuticals produce pioneering and innovative treatments, medicines and vaccines in the veterinary industry. When they turned their attention to the creation of a bacteriophage cocktail to prevent Salmonella infections in farm animals, they contacted us to assist with increasing their bacteriophage production.

Sales Director, Craig Loftus, visited their lab in Poland with the CellMaker Regular controller and 8L CellMaker Enclosure to demonstrate the capabilities of our machine. They had been producing S. Enteriditis at a rate of 5×1010 FPU. Craig ran their usual process using the CellMaker to compare results.

The disposable CellMaker Bag was filled with 5L of LB cultivation media and pre-warmed to 37°C using our built-in, automated heater cooler system. The media was then inoculated with overnight culture of S. enterica serovar Enteritidis strain. The bioreactor was supplied continuously by 5 litres of air per minute and the temperature was stable at 37°C. Optical density of growing bacteria was measured every 30 minutes and after 2 hours it reached OD600 0.4 – 0.5 and the inoculum with bacteriophages, specific for Salmonella, in amount of 5×1010 PFU (Plaque Forming Units) was added. The culture was sustained for another 3 hours to allow the bacteriophages to amplify.

Samples were taken to measure titre of bacteriophages (in PFU/ml) and optical density of bacteria. We also ran a control batch using the same conditions but without inclusion of bacteriophages to compare the growth of bacteria.

The kinetics of optical density of bacteria growing alone and with bacteriophages was compared. Please note the comparison in the graph below. The dashed line represents bacteria growing alone while the continuous navy line shows the changing optical density when phages were added to the culture after 2 hours. The orange line represents the growing number of bacteriophages.

These results show the CellMaker successfully cultivated S. Enteritidis and further high amplification efficiency of bacteriophages.

Proteon concluded:

“The Cellexus CellMaker is a user friendly ready to use system for production of bacteriophage, and the single-use CellMaker Bag, that does not need to be sterilized prior to cultivation of bacteria, is a great alternative to traditional bioreactors. The integrated control software allows for continuous control over the conditions in the bioreactor and samples can be taken from the culture during cultivation without pausing the system. The results prove that the Cellexus CellMaker bioreactor system can be successfully used for cultivation of Salmonella enterica serovar Enteritidis and further high amplification efficiency of bacteriophages specific against these bacteria. Since, additional optimization of the process has been carried out to improve the yield of bacteriophages and is now used as production process.”

 

Data was kindly provided by Justyna Klimczak and Arkadiusz Wojtasik (Proteon Pharmaceuticals SA).