Microscale Perfusion-Based Cultivation forPichia pastorisClone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes

Pichia pastorishas emerged in the past years as a promising host for recombinant protein and biopharmaceutical production. In the establishment of high cell density fed-batch biomanufacturing, screening phase and early bioprocess development (based on microplates and shake flasks) still represent a bottleneck due to high-cost and time-consuming procedures as well as low experiment complexity. In the present work, a screening protocol developed forP. pastorisclone selection is implemented in a multiplexed microfluidic device with 15 mu L cultivation chambers able to operate in perfusion mode and monitor dissolved oxygen content in the culture in a non-invasive way. The setup allowed us to establish carbon-limited conditions and evaluate strain responses to different input variables. Results from micro-scale perfusion cultures are then compared with 1L fed-batch fermentation. The best producer in terms of titer and productivity is rapidly identified after 12 h from inoculation and the results confirmed by lab-scale fermentation. Moreover, the physiological analyses of the strains under different conditions suggested how more complex experimental conditions are achievable despite the relatively easy, straight-forward, and cost-effective experimental setup. Implementation and standardization of these micro-scale protocols could reduce the demand for lab-scale bioreactor cultivations thus accelerating the development of protein production processes.

Automated summary

Pichia pastoris has become a promising host for recombinant protein production. A screening protocol for P. pastoris clone selection was developed and implemented in a multiplexed microfluidic device, allowing for rapid identification of the best producer strain. The study suggests that despite the relatively simple, straightforward, and cost-effective experimental setup, more complex experimental conditions can be achieved.