an oxygen limitation during day five of cultivation. Interestingly, this is the time immediately before achieving the maximum living cell density, which was again 1.6 x 10 cell mL-1. Independent on the experiment series, all on-line pH data corresponded well with those of off-line measured samples. 7
Fig. 2 Experimental and model-derived kLa values in investigated PreSens' flasks. Each line indicated with numbers from 20 to 150 complies with a constant filling level given in mL.
Oxygen transfer studies during mass propagation
Mass propagations of Sf-9 and Sf-21 cells were performed in 250 mL shake flasks equipped with integrated and pre-calibrated pH and DO sensors from PreSens. They lasted between seven and eight days while the flasks were filled with 100 mL Sf-900 III SFM, inoculated with 5 x 10 cells mL-1 and incubated at 27 °C and 110 rpm. Under these conditions an oxygen transfer coefficient of 5.6 per hour can be assumed. Sampling was done in two different ways. In experiment series 1 (Sf-9 cell mass propagation) each day of cultivation a sample of all flasks was taken, whereas in exeriment series 2 (Sf-21 cell mass propagation) a sample of only one shake flask was taken per day. The sample analyses included measurements of living cell density and viability (with NucleoCounter from Chemometec and Cedex HiRes from Innovatis), and off-line determinations of pH, glucose, lactate, ammonium, glutamine, glutamate (with BioProfile 100 from Nova Biomedical). 5
Fig. 3 shows a DO course which is influenced by sample taking respectively opening the sterile closure such as observed in experiment series 1, where a maximum living cell density of 1.6 x 107 cells in mL-1 was reached at day six of cultivation. However, no clear statement on possible mass transfer limitations can be made.
Fig. 3 (A) Growth course of Sf-9 suspension cells in 250 mL PreSens' flasks (experiment series 1). (B) Corresponding DO course.
Our results reveal PreSens' shake flasks, allowing non-invasive pH and DO measurement, advantageous usage for process characterization and optimized processing in case of insect cell cultivations at small scale. Oxygen is the key element for high cell density growth aimed in seed train productions and high product titers desired in protein productions.
From results of experiment series 2 in which a non-influenced DO course could be guaranteed it is obvious that cell growth of Sf-21 suspension cells stops below oxygen saturation of 10 % . Because of the fact that this observation coincides with those of further groups and growth inhibitions resulting from culture medium appear implausible to our experience, we suppose
 Ries C, John G, John C, Eibl R, Eibl D (2009) A shaken disposable bioreactor system for controlled insect cell cultivations at millilitre-scale. Life Science Engineering, submitted.