Effect of CO 2 on Uninfected Sf-9 Cell Growth and Metabolism Sucheta G. Vajrala and David W. Murhammer Department of Chemical and Biochemical Engineering, 4133 Seamans Center, The University of Iowa, Iowa City, IA 52242 DOI 10.1002/btpr.2229 Published online February 9, 2016 in Wiley Online Library (wileyonlinelibrary.com) A problem in the mass production of recombinant proteins and biopesticides using insect cell culture is CO 2 accumulation. This research investigated the effect of elevated CO 2 con- centration on insect cell growth and metabolism. Spodoptera frugiperda Sf-9 insect cells were grown at 20% air saturation, 27 8 C, and a pH of 6.2. The cells were exposed to a con- stant CO 2 concentration by purging the medium with CO 2 and the headspace with air. The population doubling time (PDT) of Sf-9 cells increased with increasing CO 2 concentration. Specifically, the PDT for 0-37, 73, 147, 183, and 220 mm Hg CO 2 concentrations were 23.2 6 6.7, 32.4 6 7.2, 38.1 6 13.3, 42.9 6 5.4, and 69.3 6 35.9 h (n 5 3 or 4, 95% confi- dence level), respectively. The viability of cells in all experiments was above 90%, i.e., while increased CO 2 concentrations inhibited cell growth, it did not affect cell viability. The osmo- lality for all bioreactor experiments was observed to be 300–360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO 2 concentration did not significantly alter the cell specific glucose consumption rate (2.5–3.2 3 10 217 mol/cell s), but slightly increased the specific lactate production rate from 23.0 3 10 219 to 10.2 3 10 219 mol/cell s. Oxidative stress did not contribute to CO 2 inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl con- centrations was discovered at elevated CO 2 concentration. V C 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:465–469, 2016 Keywords: Spodoptera frugiperda Sf-9 cells, carbon dioxide inhibition, metabolism, cell growth Introduction The prominence of insect cell culture has grown rapidly in recent years due to its ability to produce baculovirus biopes- ticides and recombinant proteins using insect cell culture. Large-scale production of these products involves high-cell- density bioreactors with large working volumes leading to the accumulation of CO 2 to inhibitory levels within the bio- reactor. The use of small bubbles to provide sufficient dis- solved oxygen to the shear sensitive insect and mammalian cells added to the increased dissolved CO 2 concentrations in the cell culture medium. 1–3 Furthermore, in large-scale bio- reactors, the surface-to-volume ratio decreases with increas- ing bioreactor size to exacerbate CO 2 accumulation within the bioreactor. As CO 2 accumulates in the bioreactor, it dissolves in the cell culture medium and enters the cell cytoplasm (i.e., CO 2 passes freely across cell membranes), where it dissociates into HCO 2 3 and H 1 (Eq. 1). CO 2 1 H 2 O $ H 1 1 HCO 3 2 (1) Using Henry’s law, the equation given by Sperandio and Paul 4 and assuming that the medium ionic strength is 0.35 M (corresponding to a typical osmolarity of 0.35 M for insect cell culture medium), the solubility of CO 2 in insect cell culture medium can be estimated as 25.0 mm Hg/mM. 5 The CO 2 concentration that is typically required for nor- mal cell growth is 30–50 mm Hg, whereas CO 2 concentra- tions above 100 mm Hg inhibit cell growth and productivity. 6 Numerous investigations conducted on CHO, hybridoma, and BHK cells demonstrated that CO 2 accumula- tion decreases cell growth, viability, and product forma- tion. 6–9 Nevertheless, very few studies have been conducted to investigate the effect of elevated CO 2 concentration on insect cells. In baculovirus-infected Sf-9 cells, the quantity of recombinant protein produced in a 150 L bioreactor was only 30% of that produced in the 70 mL spinner flask and this was believed to be due to CO 2 accumulation. 10 How- ever, no actual CO 2 measurements were taken, but a mathe- matical model predicted that CO 2 levels reached a peak value of 114 mm Hg. Experiments conducted in 100 mL spinner flasks demonstrated that the production of b-gal and TGFb recombinant proteins at 15% CO 2 were reduced by 39% and 56%, respectively, compared to the production at 0% CO 2 . In addition, extended protein production occurred in the 15% CO 2 environment due to slower cell death. This was hypothesized to be due to a delayed infection process resulting from cell metabolism inhibition. Contract grant sponsor: National Science Foundation; Contract grant number: 0541948. Current address of S.G. Vajrala: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878. Resubmitted for publication in Biotechnology Progress on October 14, 2015. Correspondence concerning this article should be addressed to D.W. Murhammer at david-murhammer@uiowa.edu. V C 2016 American Institute of Chemical Engineers 465