Small-scale development and optimization of stirred tank mammalian cell perfusion cultures
September 17-21, 2017
The continuous production of biopharmaceuticals combines several advantages: the use of smaller equipment, faster and more efficient processing, higher volumetric productivities, as well as a more uniform product quality . Mammalian cell perfusion cultures employing an external cell retention device have shown to support long-term operation at very high viable cell densities with the possibility of direct integration to subsequent downstream steps  However, knowledge on time and cost effective development and scale-up procedures to achieve a reliable reactor operation and desired product characteristics is still limited. Therefore, this study aims at the definition of a comprehensive optimization framework for mammalian cell cultures utilizing a combination of small-scale spin tube and bench top bioreactor experiments. In a first step, rapid evaluation of suitable operating conditions in small scale simulated perfusion cultures allowed the identification of key process parameters to facilitate steady state operation and process control. Their choice was further refined in a stirred tank perfusion bioreactor setup  by sequential screening of steady states while targeting improved product yields and desired product quality characteristics. Varying one parameter at a time, measurements of extra- and intracellular metabolites, product concentration and quality attributes were used to optimize the reactor performance. Comparable growth behaviour and metabolite consumption/production rates were observed between experimental scales. The tuning of key cell culture parameters led to a superior performance of the perfusion culture. Especially, the decrease of the cell specific perfusion rate (CSPR) prevented excessive cellular growth and significantly reduced the loss of product in the bleed stream. Constant patterns of product quality attributes such as N-linked glycosylation and charge isoforms were observed within each steady state but slight varied between different operating points. Overall, this study underlines the potential of perfusion cultures to simultaneously achieve high productivities while tuning towards desired characteristic of consistently expressed therapeutic proteins. Literature  V. Warikoo, R. Godawat, K. Brower, S. Jain, D. Cummings, E. Simons, T. Johnson, J. Walther, M. Yu, B. Wright, J. Mclarty, K. P. Karey, C. Hwang, W. Zhou, F. Riske, and K. Konstantinov, “Integrated continuous production of recombinant therapeutic proteins,” Biotechnol. Bioeng., vol. 109, no. 12, pp. 3018–3029, 2012.  R. Godawat, K. Konstantinov, M. Rohani, and V. Warikoo, “End-to-end integrated fully continuous production of recombinant monoclonal antibodies,” J. Biotechnol., vol. 213, pp. 13–19, 2015.  D. J. Karst, E. Serra, T. K. Villiger, M. Soos, and M. Morbidelli, “Characterization and comparison of ATF and TFF in stirred bioreactors for continuous mammalian cell culture processes,” Biochem. Eng. J., vol. 110, pp. 17–26, 2016.
Moritz Wolf, Massimo Morbidelli, and Daniel Karst, "Small-scale development and optimization of stirred tank mammalian cell perfusion cultures" in "Integrated Continuous Biomanufacturing III", Suzanne Farid, University College London, United Kingdom Chetan Goudar, Amgen, USA Paula Alves, IBET, Portugal Veena Warikoo, Axcella Health, Inc., USA Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/biomanufact_iii/41
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