P35: PichiaPro- providing automated, advanced, process control for Pichia pastoris fermentation

The ability to conduct fully automated advanced fermentation strategies for Pichia pastoris on routine bench top equipment would be a powerful platform for process optimization.  Maximizing AOX1 driven product yields during fermentation involves achieving a balanced cellular metabolic state, ensuring productive translation of abundant transcript.   However, establishing long-term control over culture metabolism is non-trivial as cultures are dynamic systems.  AOX1 induction levels and cellular metabolic state are most directly influenced by culture growth rate and the carbon source ratio (methanol:glycerol) provided to meet growth rate targets.  Advanced fermentation methods utilize dynamically calculated solutions to mathematical models of culture metabolism to achieve set point growth rate control.   Such methods are common in specialized labs, but not widely available.

We have implemented a growth model based fermentation control software, we name “PichiaPro”, that runs within the BioCommand software common to nearly all fermentation systems produced by New Brunswick (an Eppendorf Company).  PichiaPro enables growth rate control and mixed production feeds based upon dynamic solutions to a common model of cellular growth.  PichiaPro fully automates these methods, while achieving excellent process stability and run-to-run consistency.

Using a tdTomato expressing strain we demonstrate nearly exact correlation between culture growth rate set point and observed values over extended production times.  This holds true across a range of conditions covering feed ratios from (0:1)->(2:1) (glycerol:methanol) during production.  PichiaPro executes the entire fermentation process automatically, runs on widely available bench top fermentation systems, and enables rapid searching of metabolic parameter space in optimization of product yields.