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

Damiano Totaro, Bojana Radoman, Bernhard Schmelzer, Mario Rothbauer, Matthias G Steiger, Torsten Mayr, Michael Sauer, Peter Ertl, Diethard Mattanovich

Biotechnology journal 2021 Mar

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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. © 2020 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.

Citation

Damiano Totaro, Bojana Radoman, Bernhard Schmelzer, Mario Rothbauer, Matthias G Steiger, Torsten Mayr, Michael Sauer, Peter Ertl, Diethard Mattanovich. Microscale Perfusion-Based Cultivation for Pichia pastoris Clone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes. Biotechnology journal. 2021 Mar;16(3):e2000215