Abstract
Current CHO cell production processes require an optimized space-time-yield. Process intensification can support achieving this by enhancing the productivity and improving facility utilization. The use of perfusion at the last stage of the seed train (N-1) for high cell density inoculation of the fed-batch N-stage production culture is a relatively new approach with few industry applicable examples. Within this work, the impact of the cell-specific perfusion rate (CSPR) of the N-1 perfusion and the relevance of its control for the quality of generated inoculation cells was evaluated using an automated perfusion rate (PR) control based on online biomass measurements. Precise correlations (R² = 0.99) between permittivity and viable cell counts were found up to the high densities of 100⋅106 c·mL−1. Cells from N-1 perfusion were cultivated at a high and low CSPR with 50 and 20 pL·(c·d)−1, respectively. Lowered cell growth and an increased apoptotic reaction was found as a consequence of the latter due to nutrient limitations and reduced uptake rates. Subsequently, batch cultivations (N-stage) from the different N-1 sources were inoculated to evaluate the physiological state of the inoculum. Successive responses resulting from the respective N-1 condition were uncovered. While cell growth and productivity of approaches inoculated from high CSPR and a conventional seed were comparable, low CSPR inoculation suffered significantly in terms of reduced initial cell growth and impaired viability. This study underlines the importance to determine the CSPR for the design and implementation of an N-1 perfusion process in order to achieve the desired performance at the crucial production stage.
Highlights
Mammalian cell lines are the predominantly used host for the pro duction of different recombinant therapeutic proteins (Jayapal et al, 2007) primarily being monoclonal antibodies (Kunert and Reinhart, 2016)
This study demonstrates that process intensification at the N-1 stage can be effectively achieved by implementing perfusion
viable cell concentration (VCC) of up to 100⋅106 c⋅mL− 1 were obtained by using online biomass measurements to control the perfusion rate (PR) accurately based on a cell-specific perfusion rate (CSPR) setpoint
Summary
Mammalian cell lines are the predominantly used host for the pro duction of different recombinant therapeutic proteins (Jayapal et al, 2007) primarily being monoclonal antibodies (mAbs) (Kunert and Reinhart, 2016). Continuous biomanufacturing applying pro cess intensification of upstream processes has received much attention due to its ability to further optimize current state-of-the art mAb pro duction capabilities (Bielser et al, 2018; Karst et al, 2018). At its end (N-1 stage) cells are sufficiently propagated for inoculation of the production bioreactor (N-stage). Fed-batch (FB) processes in stirred tank reactors are pre dominantly used in the biomanufacturing industry at the N-stage up to m3-scales (Ecker and Ransohoff, 2014; Kantardjieff and Zhou, 2014).
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