TY - JOUR
T1 - Microscale Perfusion-Based Cultivation for Pichia pastoris Clone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes
AU - Totaro, Damiano
AU - Radoman, Bojana
AU - Schmelzer, Bernhard
AU - Rothbauer, Mario
AU - Steiger, Matthias G.
AU - Mayr, Torsten
AU - Sauer, Michael
AU - Ertl, Peter
AU - Mattanovich, Diethard
PY - 2021/3
Y1 - 2021/3
N2 - 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.
AB - 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.
KW - bioprocess development
KW - fed-batch fermentation
KW - Pichia pastoris
KW - recombinant protein production
KW - screening phase
UR - http://www.scopus.com/inward/record.url?scp=85092139762&partnerID=8YFLogxK
U2 - 10.1002/biot.202000215
DO - 10.1002/biot.202000215
M3 - Article
C2 - 32935449
AN - SCOPUS:85092139762
SN - 1860-6768
VL - 16
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 3
M1 - 2000215
ER -