Microscale Perfusion‐Based Cultivation for Pichia pastoris Clone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes

Abstract Pichia pastoris has 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 for P. pastoris clone selection is implemented in a multiplexed microfluidic device with 15 μL 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.