High cell density growth of High Five suspension cells in DO-controlled wave-mixed bioreactors

1. High cell density growth of High Five suspension cells in DO-controlled wave-mixed bioreactors Teddy Beltrametti1, Nicole C. Bögli1, Gerhard Greller2, Regine Eibl1, Dieter Eibl1 1 Zurich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Biotechnology, Biochemical Engineering and Cell Cultivation Technique, nicole.boegli@zhaw.ch; Grüental, CH-8820 Wädenswil, www.lsfm.zhaw.ch, www.bioverfahrenstechnik.ch and www.zellkulturtechnik.ch 2 Sartorius Stedim Biotech GmbH, August-Spindler-Str. 11, D-37079 Goettingen, www.sartorius-stedim.com Background and motivation In addition to Sf-9 cells from Spodoptera frugiperda, High Five™ (Hi-5) cells represent the most often used host cells in baculovirus expression vector system (BEVS) based processes aimed at recombinant proteins [1]. In serum-free culture mediums and batch modes non-infected Hi-5 cells already grow to maximum cell densities of between 8 - 9 x 106 cells mL-1 while reaching doubling times of between 18 and 29 hours. In fact, they can even achieving higher specific protein production rates than Sf-9 cells [2]. Preconditions are an optimum cultivation temperature of between 27 and 28 °C, no oxygen limitation, an optimum pH-value of approximately 6.3 and protection for the cells against shear stress and strong foaming in dynamic cultivation systems [3, 4]. Whereas Weber and Fussenegger [4] and Ries et al. [5] have shown advantageous growth (homogeneous energy dissipation, negligible foaming) of Sf-9 suspension cells in the disposable BioWave and its successor, the BIOSTAT CultiBag RM, no references for cultivation of Hi-5 cells in wave-mixed bioreactors were found. Therefore investigations were carried out to determine the process control mode that achieves maximum cell densities for Hi-5 cells in the BIOSTAT CultiBag RM optical, operating in batch mode with 1 L culture volume. Finally, the possibility of cell growth improvement by applying pH or/and DO control strategies was focused on. Material and methods B A All experiments were performed in the BIOSTAT CultiBag RM 20 optical from Sartorius Stedim Biotech (Fig. 1A) with integrated optical pH and DO sensors. The sensor systems consisting of a reusable light fibre cable and fluorescent indicator were discarded with the bag. A tube guide supports installation of the light fibre cable (Fig. 1B). In all growth trials, Hi-5 suspensions cells and Express Five SFM from Gibco Invitrogen were used. Seed inoculum was generated in single-use shake flasks (Corning) and incubated in an Infors`Ecotron shaker (27 °C, 100 rpm, 25 mm shaking diameter). The starting parameters for the BIOSTAT CultiBag RM optical were: 27 °C, 0.2 vvm (sterile filtered air), 6 ° rocking angle and 20 rpm rocking rate. Inoculation density was always 1 x 106 viable cells mL-1, whereby the BIOSTAT CultiBag RM optical was operated as follows: non-controlled (only pH- and DO monitored), DO controlled (DO set point 50 %), pH controlled (set point of 6.3, adjustment of 1 M potassium base) as well as pH and DO controlled. Each control strategy experiment was performed at least twice. DO control was realized with a cascade by increasing the rocking rate up to 28 rpm. If required, pure oxygen was added. In addition to the on-line measurements, daily sampling was carried out for off-line determination of pH-value, cell density, viability as well as nutrient and metabolite concentrations (glucose, glutamine, lactate, ammonium). B www.sartorius-stedim.com Figure 1: (A) Seed inoculum production with the Biostat CultiBag RM 20 optical; (B) Tube guide and cap with fluorescent indicator. Results and discussion In Table 1 maximum values of viable cell density [xmax], growth rate [µmax] and doubling time [td,max] are shown. The maximum viable cell density ranged of between 6 - 9.35 x 106 cells mL-1 and was reached in 43 to 67.5 hours post inoculation in the DO controlled mode (see also Fig. 2A, blue line). Interestingly, the maximum viable cell density was achieved up to 24 hours earlier than for the uncontrolled and pH controlled modes. While the DO set point (50 %) remained constant in the controlled experiments, those in uncontrolled mode decreased after the maximum cell density to 10 % (Fig. 2B). As can be clearly seen in Fig. 2C the pH-values were of between 5 and 7.5, indicating that pH-value control is redundant. The highest lactate accumulation (2.5 - 4.1 g L-1) was witnessed in the non-controlled and pH controlled runs, in which also the lowest maximum viable cell densities were measured. The glucose was completely consumed by the end of all the experiments. Comparable experiments from Riehl et al. [3] performed in standard stirred cell culture bioreactors were characterized by similar maximum cell densities to the BIOSTAT CultiBag RM. Tabel 1: Reached viable cell densities, growht rates and doubling times. BIOCHEMICAL ENGINEERING AND CELL CULTIVATION TECHNIQUE However, maximum growth rates (0.028 h-1) in the stirred bioreactor were 35 % lower than in the BIOSTAT CultiBag RM. This finding was also seen in the stirred Mobius CellReady 3 L (Merck Millipore), where the maximum growth rate was 14 % lower than in the BIOSTAT CultiBag RM and a strong foaming despite the addition of an antifoam agent. Conclusions For Hi-5 suspension cells, DO controlled operation of the BIOSTAT CultiBag RM optical (set point 50 %) is achieved 35 % higher maximum living cell densities in comparison to the uncontrolled mode. As shown in Figure 2C, on-line control of pH-value works reliably and allows early detection of complete glucose consumption. An additional pH control with 1 M potassium base resulted in no significant growth improvement. Consequently, the BIOSTAT CultiBag RM optical (pH on-line monitoring and DO-control) should be preferred for optimum seed inoculum productions with Hi-5 suspension cells. Here even the addition of acid for pH control and the use of an antifoam agent are no longer required [6]. Figure 2: Growth experiments with Hi-5 suspension cells in the BIOSTAT CultiBag RM20 optical, uncontrolled (black), DO control (blue), pH and DO control (grey). (A) Growth and viability courses independent of control strategy: ● Viable cell density, ∆ viability; (B) Courses of DO (-) in dependence on control strategy; (C) on-line pH measurements (+) in dependence on control strategy. Values in graphs A are average values including the standard deviation of experiment couples with comparable strategy of control. Values in graphs B and C represent the typical course of one experiment. References: [1] Durocher Y., Butler M. (2009): Expression systems for therapeutic glycoprotein production. Current opinion in biotechnology 20:700-707. [2]Benslimane C., Elias B. C., Hawari J., Kamen A. (2005): Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabling studies. Biotechnology progress 21:78-86. [3]Rhiel M., Mitchell-Logean C. M., Murhammer D. W. (1997): Comparison of Trichoplusia ni BTI-Tn-5B1-4 (High Five™) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. Biotechnology and bioengineering 55:909-920 [4] Weber W., Fussenegger M. (2009): Insect cell-based recombinant protein production. Cell and tissue reaction engineering: Principles and practice 263-277. Berlin/Heidelberg, Springer-Verlag. [5] Ries C., John C., Eibl R. (2011):A new scale-down approach for the rapid development of Sf21/BEVS-based processes - a case study, Single-use technology in biopharmaceutical manufacture 208-213. John Wiley & Sons, Hoboken, New Jersey. [6] Beltrametti T., Bögli N. C., Ries C., Greller G., Eibl R., Eibl D. (2011): Zellkultivierung in einem wellendurchmischten, DO-regulierten Einwegbioreaktor 22-23. BIOForum 1. Contact: ZHAW Institute of Biotechnology, Wädenswil Nicole Bögli nicole.boegli@zhaw.ch Sartorius Stedim Biotech GmbH, Goettingen Gerhard Greller gerhard.greller@sartorius-stedim.com