Infoscience

Thesis

OrbShake Bioreactors for Mammalian Cell Cultures: Engineering and Scale-up

Most of the biopharmaceuticals nowadays are produced by animal cells. The worldwide demand for a specific group of biopharmaceuticals, the monoclonal antibodies (mAbs), has significantly increased this last decade. The mAbs are used as therapeutics in particular against cancer and autoimmune disorders. Most of the mAbs are produced as recombinant proteins by mammalian cells which ensure human-like post-translationnal modifications to obtain a fully functional product. However, bioprocesses with mammalian cells are usually expensive and the mAbs are needed in large amounts. As a consequence, highly flexible and cost-effective bioprocesses are required for new drug research and development as well as for the production of approved biopharmaceuticals. Orbitally shaken bioreactors (OSRs) are based on the disposable technology (single-use bioreactors) and are therefore very flexible. This work aims to improve our understanding of these bioreactors by studying their engineering principles such as mixing, the gas transfer and the hydrodynamics in the frame of mammalian cell culture. The mixing study showed that OSRs ensure homogeneity of the mammalian cell culture at scales up to 1500 L with mixing times below 30 s. By keeping the geometric ratios (shaking diameter, inner diameter and liquid height) and the Froude number constant, mixing and the free-surface shape of a 1500-L OSR could be mimicked in a 30-L OSR. A minimal volumetric mass transfer coefficient of oxygen (kLa) was required to operate mammalian cell culture without needing controllers to compensate the pH or the dissolved oxygen concentration (DO). This minimal kLa was cell-line dependent and typically in the range of 7-10 h-1. Cultures with the same kLa showed the same growth characteristics, recombinant protein concentrations, culture conditions (pH and DO), and metabolite profiles. A probe-independent bioprocess was run in a 200-L OSR at the same kLa as 1- and 5-L OSRs. Similar cell growth, recombinant protein concentration, pH and DO profiles were observed in the three OSRs. The shear stress level calculated by computational fluid dynamics (CFD) in a 1-L OSR at 110 rpm was one to two orders of magnitude lower than the one reported to damage mammalian cells. The CFD simulations showed that the maximal shear stress in OSRs was located at the tip of the wave, while most of the zones in the liquid had shear stress values below or equal to 0.075 Pa. Cell damage in a 1-L OSR was recorded at agitation rates ≥ 170 rpm which corresponded to maximal shear stress values of 0.2 Pa. Probe-independent bioprocesses in OSRs were compared side-by-side with fully controlled bioprocesses in stirred-tank bioreactors (STRs). The bioprocesses in OSRs were as efficient as those in STRs. Furthermore, a very low variability (≤ 10% for cell growth, recombinant protein concentration, pH and DO profiles) was observed for probe-independent bioprocesses in 5-L OSRs. Together, the results presented in this thesis demonstrate that probe-independent bioprocesses are scalable in OSRs from 50 mL to 100 L.

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