Insect Cell Cultivation in Orbitally Shaken Flasks with Sensors
Oxygen mass transfer in insect cell-based high cell density cultivations
N. Riesen, C. Ries, R. Eibl, D. Eibl Zurich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Biotechnology, Biochemical Engineering and Cell Cultivation Techniques, Wädenswil, Switzerland
Insect cell-based processes have become increasingly influential for tool protein and Virus-like Particle (VLP) vaccine productions over the last years. At small scale insect cells are normally grown in non-instrumented orbitally shaken disposable flasks, in most cases Erlenmeyers, in order to perform seed train productions and early stage process developments. The Shake Flask Reader (SFR) with non-invasive optical sensors for pH and DO is an important contribution to easy quantification of oxygen mass transfer in Erlenmeyers recently made. In addition, this allows controlled processing at mL-scale as exemplified for described Sf-9 and Sf-21 cell mass propagation procedures.
Insect cells and their characteristics
Since the early 1970s the suitability of insect cells (in conjunction with the Baculovirus Expression Vector System, BEVS) for production of complex proteins used in structural analyses, functional studies, manufacture of diagnostics and vaccines have been described in numerous articles and reviews. The applied production cell lines (Sf-9, Sf-21 and BTI-TN-5B1-4, also known as High FiveTM or Trichoplusia ni cell line) derive from the Fall Armyworm (Spodoptera frugiperda) and the Cabbage Looper (Trichoplusia ni). Even in batch mode and at temperatures between 27 °C and 28 °C, and a pH value ranging between 6.2 and 6.9 these cells grow up to high cell densities (>1 x 107 cells mL-1). For this it is of course necessary that an optimized serum-free culture medium exists, which supports cell growth as well as a qualified cultivation system preventing shear stress damage and guaranteeing sufficient oxygen supply. The parameter characterizing the oxygen transfer in a cultivation system is the kLa value (also known as oxygen transfer coefficient).
Determination of kLa values
In order to determine kLa values for shaken flasks from PreSens (250 mL total volume, standard vented caps), their coupling with PreSens' SFR (www.presens.de/SFR) is required. The SFR unit mounted on Infors' shaking incubator Ecotron (25 mm shaking diameter) has nine double optical modules which enable wireless real-time monitoring and display of pH and DO. By using the well- known standard gassing-out method (see Fig. 1), nine different experiments (statistically designed with STAVEX 5.0 software) at varying filling volumes (between 25 - 150 mL) as well as shaking frequencies (between 80 - 200 rpm) were carried out. All flasks were running with Sf-900 III SFM from Gibco Invitrogen at a temperature of 27 °C.
Fig. 1 Experimental set-up for the determination of kLa values. (A) gassing-out with N L b y a s i l i c o n e t u b e i n s e r t e d t h r o u g h a h o l e i n t h e v e n t e d c a p ( B ) . A f t e r p u r g i n g w i t h N 2 t h e s i l i c o n e t u b e ( C ) w a s w i t h d r a w n i n o r d e r t o r e p l a c e t h e h e a d s p a c e w i t h a i r .
DO concentrations were monitored over the time and experimental as well as STAVEX 5.0 model derived kLa values were calculated as described in detail by Ries et al. . At selected cultivation parameters experimental kLa- values ranged between 4.4 and 37.9 per hour; these values correspond well with the modeled oxygen transfer data (see Fig. 2).