Experimental studies from shake flasks to 3 L stirred tank bioreactor of nutrients and oxygen supply conditions to improve the growth of the avian cell line DuckCelt®-T17

Abstract

Background: To produce viral vaccines, avian cell lines are interesting alternatives to replace the egg-derived processes for viruses that do not grow well on mammalian cells. The avian suspension cell line DuckCelt^®-T17 was previously studied and investigated to produce a live attenuated metapneumovirus (hMPV)/respiratory syncytial virus (RSV) and influenza virus vaccines. However, a better understanding of its culture process is necessary for an efficient production of viral particles in bioreactors.

Results: The growth and metabolic requirements of the avian cell line DuckCelt^®-T17 were investigated to improve its cultivation parameters. Several nutrient supplementation strategies were studied in shake flasks highlighting the interest of (i) replacing L-glutamine by glutamax as main nutrient or (ii) adding these two nutrients in the serum-free growth medium in a fed-batch strategy. The scale-up in a 3 L bioreactor was successful for these types of strategies confirming their efficiencies in improving the cells' growth and viability. Moreover, a perfusion feasibility test allowed to achieve up to ~ 3 times the maximum number of viable cells obtained with the batch or fed-batch strategies. Finally, a strong oxygen supply - 50% dO₂ - had a deleterious effect on DuckCelt^®-T17 viability, certainly because of the greater hydrodynamic stress imposed.

Conclusions: The culture process using glutamax supplementation with a batch or a fed-batch strategy was successfully scaled-up to 3 L bioreactor. In addition, perfusion appeared as a very promising culture process for subsequent continuous virus harvesting.

Keywords: Cell growth; DuckCelt®-T17 avian cell line; Fed-batch culture; Glutamax; Nutrient supplementation.

Fig. 1

Characterization of the DuckCelt®-T17 culture process performed using reference operating conditions from [20] in a 3 L stirred bioreactor. Time evolution of cell growth (solid line) and viability percentage (dotted line) (a), of the concentration of metabolites involved in glutaminolysis (b) and glycolysis (c). Gln: glutamine (solid line); NH₄⁺: ammonium (dotted line); Glc: glucose (solid line); Lac: lactate (dotted line). Results are presented as means ± SD (n = 3)

Fig. 2

Effect of dO₂ on cell growth (a), viability (b) and growth kinetic parameters (c) for DuckCelt®-T17 cells cultivated in a 3 L bioreactor. The cells were grown at 10% (grey line), 30% (blue line) or 50% (green line) dO₂. Results are presented as means ± SD (n = 3 for 30% and n = 2 for 10% dO₂). The data for 50% dO₂ (n = 3) originate from the reference process experiments

Fig. 3

Effect of the substitution of glutamine (strategy A) by glutamax (strategy B) in the culture medium for the DuckCelt®-T17 cell culture in shake flasks. Time evolution of cell growth (solid line) and viability percentage (dotted line) (a), of the concentration of metabolites involved in glutaminolysis (b) and glycolysis (c). Glutamine and glucose are represented in solid line and lactate and ammonium in dotted line. Results are presented as means ± SD (n = 6 for strategies A and B, n = 2 for strategy C (without glutamine))

Fig. 4

Effect of various supplementation strategies on the maximal concentration of viable cells (VCC_max) characterizing the DuckCelt®-T17 growth in shake flasks. Results are presented as means ± SD (n = 6 for strategies A, B, D, E and J, n = 3 for the other supplementations conditions). Asterisks (*) and (**) indicate p-value < 0.05 and 0.01, respectively with Student’s t-test

Fig. 5

Effect of medium supplementation strategies D, E, H, I and J combining glutamine and/or glutamax as compared to the reference strategy A in shake flasks. Time evolution of glutamine consumption (a) and ammonium (b) and lactate (c) productions during cell culture. Results are presented as means ± SD (n = 6 for strategies A, D, J, n = 5 for strategy E and n = 3 for strategies H and I)

Fig. 6

Effect of mimicking fed-batch culture (strategies K and M) by adding OptiPRO™ SFM during the culture in shake flask compared to strategies A and B. Time evolution of cell growth (a), viability percentage (b) and ammonium production (c). Results are presented as means ± SD (n = 6 for strategies A and B, n = 3 for strategies K and M)

Fig. 7

Scale-up in a 3 L bioreactor of the DuckCelt®-T17 culture using batch (glutamine and glutamax), fed-batch (glutamax + SFM) or perfusion processes. Time evolution of cell growth (solid line) (a), viability percentage (dotted line) (b) and waste product concentration (ammonium in solid line and lactate in dotted line) (c). Comparison of metabolic profiles at day 9 between the culture permeate and the bioreactor culture medium (d). Results are presented as means ± SD (n = 3 for strategies A and M, n = 2 for strategy B, n = 1 for perfusion assay)