November 1-5, 2015
Mammalian cells are the preferable platform for secreted pharmaceutical proteins due to the compatibility of posttranslational modifications performed by these cells with therapeutic applications. However, to obtain a desirable quantity of these proteins, high cell concentrations are also needed. Baby hamster kidney BHK-21/C13 cells, adapted to single-cell suspension culture growth were cultivated in batch mode in a Biostat B (Sartorius AG, Germany) bioreactor with a 1,5 L working volume vessel. The temperature was controlled at 37 ºC, pH at 7.2 with CO2, agitation at 80 rpm and dissolved oxygen at 50% of air saturation. The culture medium used was IMDM/DMEM supplemented with Fetal Bovine Serum (5%), Pluronic F-68 as a shear protectant (2%) and L-glutamine 4 mM solution (2%). The bioreactor was inoculated with exponential-growing cells previously grown in T-25 and spinner flasks. Relevant data collected from this experiment is shown in Table 1. In order to achieve high cell concentration, the same BHK-21/C13 cells were cultivated in a perfusion set-up, with an internal polyester spin-filter BB-8808571 (Sartorius) with 10 µm diameter pore size attached to the impeller shaft as cell retention device. This set-up is different from the one previously used only by the presence of the internal spin-filter. To start the perfusion, before the feed and withdrawal pumps were turned on, the set-up was operated as a batch process until there were enough cells to start the continuous operation mode. Data collected from this phase was then compared with data obtained from the batch cultivation. Specific growth rates and exponential growth phase duration were similar for both experiments. However, the specific rates of glucose (qGLU) and glutamine (qGLN) consumption were 84% and 32% higher (Table 1), respectively, when compared to the batch cultivation. Similarly, the specific rates of lactate (qLAC) and ammonium (qNH4) formation were 78% and 102% higher, respectively. The specific rates of substrates consumption and of metabolites formation were calculated for the exponential growth phase. It is reported in literature the association of higher specific substrates consumption rates and metabolites production rates with stress factors, and we can associate the presence of the spin-filter with the physiological parameters findings, suggesting that BHK cells were exposed to a more stressful condition. The calculated value, obtained though CFD (computational fluid dynamics), for the wall shear stress of a filter rotating at 100 rpm vary from 1.57 to 1.67 Pa depending on the recirculation rate1. Despite being a small value, the system without the spin-filter already has its intrinsic shear stress value, so the stress caused by the filter must be added to and not considered as the absolute value. It was also reported that shear stress in the range from 0.75 to 1.0 Pa can affect both the viability and morphology of one BHK-21/C13 adherent cell line cultivated up to 24 hours2. Our data indicate that the presence of an internal spin-filter may be associated to cell stress.
1 FIGUEREDO-CARDERO A et al. Rotating cylindrical filters used in perfusion cultures: CFD simulations and experiments. Biotechnol Prog, v. 30, n. 5, p. 1093-1102, 2014. 2 LUDWIG A, KRETZMER G, SCHÜGERL K. Determination of a “critical shear stress level” applied to adherent mammalian cells. Enzyme Microb Technol, v. 14, n. 3, p. 209-213, 1992.