Infoscience

Thesis

Orbitally shaken bioreactors for mammalian cell culture: engineering characterization and bioprocess scale-up

Driven by the commercial success of recombinant biopharmaceuticals, there is an increasing demand for novel, cost-effective mammalian cell culture bioreactor systems for the production of biologicals requiring mammalian protein processing. Recently, orbitally shaken bioreactors at scales from a few mLs to 2'000-L have been explored for the cultivation of mammalian cells and are considered to be attractive alternatives to conventional stirred-tank bioreactors because of increased flexibility and reduced costs. Although the choice of geometry of these shaken containers is diverse with the availability of conical (i.e. shake flask), square-shaped, or cylindrical containers, the latter are preferred because more regular and predictable flow patterns are generated at all volumetric scales. The main challenge facing the scale-up of the shaken container is to maintain a moderate oxygen transfer rate (OTR) to meet the oxygen requirement of cultured cells. The volumetric mass transfer coefficient (KLa) of shaken bioreactors with working volumes from a few mLs to 1'000-L were determined, and operating parameters having an impact on the oxygen transfer were investigated. KLa values between 10-30 h-1 were typically achieved in small-scale (<1-L) shaken containers. In 200- and 2'000-L containers with a working volume of 50% of the nominal volume, moderate but satisfying KLa values of 3-8 h-1 were obtained even with mild shaking speeds. Further improvement of the OTR (2-10 fold increase of the KLa value) was achieved by introduction of special designed "mixing units" into shaken bioreactors. Using 30- and 50-L shaken cylindrical containers, the hydrodynamic properties such as the free surface shape, velocity field, and mixing behavior were analyzed by Computational Fluid Dynamics (CFD) simulations and experimental investigations. A mixing time sufficiently short for efficient cultivation of mammalian cells (less than 180 s) was observed even at very low shaking speeds. A simple mixing model with a well-mixed zone that included the liquid near the free surface and the vessel wall and a poor-mixed zone in the middle of the bulk liquid was derived. All the results obtained in cell-free studies served as a knowledge base for a direct scale-up of CHO cells in non-instrumented culture processes using bench- (<1-L) and large-scale (2'000-L) containers in a process development cycle with a feedback loop formed by scale-up and scale-down studies. In the 2'000-L shaken bioreactor at a working volume of 500 L, a maximal cell density of 6.5 x 106 cells mL-1 was achieved with a final antibody titer of 270 mg L-1 in a simple batch process. Similar cell growth profiles and productivity were obtained in parallel control cultures in 50-mL TubeSpin® bioreactors and in 1-L cylindrical bottles. These results demonstrated the feasibility of establishing a robust, standardized, and transferable technical platform for large-scale mammalian cell culture based on orbitally shaken bioreactors.

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