Process design and development of a mammalian cell perfusion culture in shake-tube and benchtop bioreactors.

The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge on time- and cost-effective procedures. The maximum achievable viable cell density (VCDmax ), minimum cell specific perfusion rate (CSPRmin ), cellular growth characteristics and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this work, we developed a stepwise procedure to use shake-tube (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg/cell/day) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 mL. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 106 cells/mL at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures. This article is protected by copyright. All rights reserved.