Adaptation for survival: phenotype and transcriptome response of CHO cells to elevated stress induced by agitation and sparging.

In this work, the response and adaption of CHO cells to hydrodynamic stress in laboratory scale bioreactors originating from agitation, sparging and their combination is studied experimentally. First, the maximum hydrodynamic stress, τ(max), is characterized over a broad range of operating conditions using a shear sensitive particulate system. Separate stress regimes are determined, where τ(max) is controlled either by sparging, agitation, or their combination. Such conditions are consequently applied during cultivations of an industrial CHO cell line to determine the cellular responses to corresponding stresses. Our results suggest that the studied CHO cell line has different threshold values and response mechanisms for hydrodynamic stress resulting from agitation or sparging, respectively. For agitation, a characteristic local minimum in viability was found after stress induction followed by viability recovery, while at highest sparging stress a monotonic decrease in viability was observed. If both stresses were combined, also both characteristic stress responses could be observed, amplifying each other. On the other hand, cellular metabolism, productivity and product quality did not change significantly. Transcriptome analysis using mRNA microarrays confirmed that separate adaptation mechanisms are activated in the different stress situations studied, allowing identification of these stresses using a transcriptome fingerprinting approach. Functional analysis of the transcripts was consequently used to improve our understanding of the molecular mechanisms of shear stress response and adaptation.

Development of a Scale-Down Model of hydrodynamic stress to study the performance of an industrial CHO cell line under simulated production scale bioreactor conditions.

The objective of this study was to develop a Scale-Down Model of a hydrodynamic stress present in large scale production bioreactors to investigate the performance of CHO cells under simulated production bioreactor conditions. Various levels of hydrodynamic stress were generated in 2L bioreactors mimicking those present in different locations of a large scale stirred tank bioreactor. In general, it was observed that tested cells are highly robust against the effect of hydrodynamic stress. However, at elevated hydrodynamic stress equivalent to an average energy dissipation rate, ε, equal to 0.4W/kg, the specific monoclonal antibody productivity, qmAb, decreased by 25% compared to the cultivation conditions corresponding to ε equal to 0.01W/kg. Even stronger decrease of qmAb, in the order of 30%, was observed when ε was periodically oscillating between 0.01 and 0.4W/kg to simulate the repeated passage of cells through the highly turbulent impeller discharge zone of a production scale bioreactor. Despite this effect, no changes in metabolite consumption or byproduct formation were observed. Furthermore, considering the experimental error product quality was independent of the applied ε. To achieve a molecular insight into the observed drop of cellular productivity, a transcriptome analysis using mRNA microarrays was performed. It was found that transcripts related to DNA damage and repair mechanisms were upregulated when high ε was applied for cultivation.