Small-scale bioreactor system for process development and optimization

An agitated 12-well microtiter plate system with a working volume of 2ml was investigated for cell culture process development. Agitation assures homogeneity in wells and enhances mass transfer between the gas and the liquid phase, thus improving maximum cell density and pH stability. The pH of the NaHCO(3)-buffered system can be adjusted by altering the carbon dioxide content of the gas phase. The non-toxic, visual pH indicator phenol red was used in combination with a spectrophotometric plate reader for rapid and precise pH measurements. For high throughputs, cell growth was assessed non-invasively using stable green fluorescent protein (GFP) expressing cells and a fluorescence plate reader. The setup is simple and inexpensive. The system can be automated and allows several hundred small-scale bioreactor experiments to be run in parallel.

Low-temperature pausing of cultivated mammalian cells

Frédéric Grosjean, David Hacker, Lisa Hunt, Martin Jordan, Florian Maria Wurm

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.

2005

OrbShake Bioreactors for Mammalian Cell Cultures

Stéphanie Tissot

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.

EPFL2011

OrbShake Bioreactors for Mammalian Cell Cultures

Stéphanie Tissot

EPFL2011