Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc.

Department of Chemistry and Applied Biosciences Institute for Chemical and Bioengineering ETH Zurich HCI F 129 Vladimir Prelog Weg 1 8093 Zurich Switzerland

Department of Chemistry and Applied Biosciences Institute for Chemical and Bioengineering ETH Zurich HCI F 129 Vladimir Prelog Weg 1 8093 Zurich Switzerland Department of Chemical Engineering University of Chemistry and Technology Technicka 3 166 28 Prague Czech Republic

Department of Chemistry and Applied Biosciences Institute for Chemical and Bioengineering ETH Zurich HCI F 129 Vladimir Prelog Weg 1 8093 Zurich Switzerland Merck Serono SA Biotech Process Sciences ZI B 1809 Corsier sur Vevey Switzerland

Merck Serono SA Biotech Process Sciences ZI B 1809 Corsier sur Vevey Switzerland

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