The rationale behind bioreactor design has been to support a continuous protein-production process. This maintains a permanent cell-free product that is capable of harvesting at high cell densities. It includes a requested combination of reliable mammalian cell retention and a highly efficient oxygen supply, as well as minimized shear stress.
For reliable cell retention, a polyethersulfone microfiltration membrane was selected and qualified. The crucial selection criteria were sharp exclusion rates with pore sizes up to 1.4-µm maximum (measured by the bubble-point method) and surface properties preventing membrane clogging over cell culture periods of 60 days.
The prerequisite for a highly efficient oxygen supply is an effective transfer rate of oxygen to every single cell in the system, even at exceptionally high cell densities. Due to low solubility of oxygen in culture media, different concepts for adequate gas supply were developed (sparger, gassing membranes). ProBioGen designed a solution that ensures a maximum distance of less than 1 mm between each cell in the system and the gas phase. Thus the ideal shape for the cell culture space is a tube.
A biomathematical model was developed at the Technical University of Hamburg-Harburg describing cell survival in such tubes. The basic assumptions of the model are an oxygen utilization rate of 1.6 pg/c*h for low-consuming cell lines (OURlow) and 6.4 pg/c*h for high-consuming ones (OURhigh). The model assumes that: the membranes are tubes filled with medium and cells, the tubes are placed in oxygen-containing gas phase, the oxygen transfer is realized only by passive diffusion.
A membrane-wall thickness of 200 µm is sufficient to support a cell density of 108 cells/mL (Figure 1) for high-consuming cell lines and a theoretical density of 109 cells/mL for low consumers inside the tube. The latter one is the cell density similar to dense human tissue.
Shear stress caused by microturbulences at the dimension of single cells is a major obstacle at high gassing and mixing rates in stirred tanks. However, the mixing of suspension cell cultures is necessary to avoid nutrient gradients. ProBioGen’s design minimizes shear stress by gentle mixing rates and sheltering the cells inside the membrane tube. In contrast to other systems, the oxygen transfer efficiency is completely independent of mixing parameters.
Identical cell culture tubes are fixed horizontally in a disposable, rotating cylindrical bioreactor vessel. Placing the cell culture tubes alternatively in oxygen or culture medium allows a sufficient nutrient supply, waste removal, and product harvest (Figure 2).
With a given length, different volumetric scales of bioreactors can be manufactured just by increasing the vessel diameter. The packaging density of the tubes stays constant.