Christian Leist

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.

Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells

CHO SSF3 cells grow as a suspension culture in unmodified commercial medium with only low–molecular weight ingredients. Continuous serum–free culture unexpectedly induced expression of a low dihydrofolate reductase activity in the originally dhfr– CHO cells. Nevertheless, it was possible with methotrexate to induce amplification of a gene coding for the hybrid plasminogen activator K2tu–PA cotransfected with a dhfr gene. Expression of K2tu–PA expression was proportionally increased to that of dhfr, which was measured with fluorescent methotrexate. Because no serum proteases were present, secreted K2tu–PA was not converted to the enzymatically active form, but was exclusively recovered in proenzyme form.

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.

Scaled-up production of recombinant human renin in CHO cells for enzymatic and X-ray structure analysis.

A process was developed to produce recombinant human renin for X-ray analysis and enzyme inhibition studies. An expression vector containing a human prorenin cDNA and expressing a mouse dihydrofolate reductase selection marker was transfected into dhfr-minus Chinese hamster ovary cells. After selection of cell strains with an increased gene copy number with methotrexate, cultures of the recombinant cells were scaled-up in serum-free media. Major improvements in cellular productivity were achieved by using continuous suspension cultures with cell recycling instead of an adherent culture system or batch-mode suspension cultures. The recombinant zymogen prorenin was purified and preparatively activated with trypsin. Enzymatic properties of the recombinant active renin are described.