Preliminary experiment in a 3 L bioreactor

Prior to our investigations, a preliminary experiment was performed to replicate the DuckCelt®-T17 cultivation process as described by Petiot et al. [20] in order to have a reference experiment. Cells were thus cultivated in a 3 L bioreactor with a 50% dO2 setpoint and 100 rpm stirring. As shown in Fig. 1a, the growth curve displayed a classical profile, with a maximal viable cell concentration (VCCmax) of about 4 × 106 cells/mL reached on day 4. The kinetic parameters showed a maximum growth rate (µmax) of 0.020 h− 1 (± 0.003 h− 1) corresponding to a doubling time (tD) of 34.7 h (± 0.4 h). The cell viability dropped below 70% from day 5, confirmed by the metabolic kinetic profiles with glucose and glutamine concentrations being close to zero, while the catabolic products lactate and ammonium rose (Fig. 1b and c). The maximal amounts of lactate and ammonium reached during the experiment were 2.8 g/L and 3.5 mM, respectively.

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)

To improve the cultivation process, two strategies were investigated in this study: (i) the modification of the dO2 parameter in the 3 L bioreactor system and (ii) the use of different supplementation strategies in both shake flasks and 3 L bioreactor.

Impact of dO2 on the growth and metabolic profiles of DuckCelt®-T17 cells in a 3 L bioreactor

Since the oxygen needs of the DuckCelt®-T17 line were unknown, the impact of dO2 on cell growth was studied at 50, 30 and 10% setpoints with stirring set at 100 rpm (Fig. 2). The growth kinetics were quite similar at 30 and 50% indicating that no O2 limitation occurred at 30%. In contrast, the cells seemed to grow significantly slower when the dO2 was set at 10%, the peak of cell growth being reached on day 8, compared to days 5 and 6 at 50 and 30%, respectively (Fig. 2a). The maximum growth rate was half as high at 10% than at 30 or 50% (Fig. 2c). Although the VCCmax was similar.

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

whatever the dO2 setpoint (about 3.5-4 × 106 viable cells/mL), viability was not. It remained stable until day 3 regardless of dO2, but while it dropped drastically at 50% dO2, it remained above 70% for a long time at 10% dO2 and at 30% dO2 it was more stable than at 50% (Fig. 2b).

A clear difference was noticed for metabolic profiles (see Figure S1 in Supplementary Information). For the 10 and 30% dO2 setpoints, glutamine was still available at day 7 contrary to 50% (Figures S1a). Ammonium stayed at acceptable levels until days 6–7 (Figures S1b). All these results drove us to set at 30% the dO2 parameter as a good compromise for subsequent experiments.

Study of various supplementation strategies in shake flasks

Given the rapid glucose depletion (see Figure S1c in Supplementary Information) and the high lactate production (data not shown), we especially investigated the effect of glutamax in our system. Indeed, the thermostable dipeptide L-alanine-L-glutamine is cleaved by the proteases produced by the cells, resulting in a sustained release of glutamine in the culture medium. We experimentally observed another advantage of glutamax compared to glutamine: a cell-free process performed for 11 days in the OptiPRO™ SFM medium exhibited spontaneous ammonium production when glutamine was used as a medium supplement while no significant ammonium production was observed with glutamax (see Figure S2 in Supplementary Information).

We compared the effect on both cell growth and metabolic profiles of the reference feeding conditions based on glutamine supplementation (strategy A) with various supplementation strategies (Table 1). Strategy B consisted in replacing glutamine by glutamax and as a control, we also performed the cell culture without glutamine (strategy C). The results in terms of cell growth and metabolic profile are shown in Fig. 3. As expected, cells did not grow without glutamine supplementation (strategy C). Strategy B induced the production of greater amounts of glutamine between day 2 and day 5 with a complete depletion observed at day 5 compared to day 4 with strategy A (Fig. 3b). Compared to strategy A, the VCCmax was significantly improved with strategy B, since 6.2 × 106 cells/mL were produced on day 5 compared to 4.7 × 106 cells/mL with the reference strategy A (Figs. 3a and 4). IVCC also increased significantly (+ 15% compared to strategy A) (Table 1). However, the kinetic parameters (µmax and tD) as well as viability were statistically similar for both conditions (Table 1). Regarding metabolites, glutamax addition had no influence on glucose depletion and ammonium production while interestingly it slightly limited the amount of produced lactate (Fig. 3b and c).

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 (VCCmax) 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

Due to its beneficial effect, glutamax was also added on day 0 (Table 1), either to the reference medium (strategy D) or to the glutamax-supplemented one (strategy E). The combination of glutamine and glutamax had obviously an important influence on the glutamine profile (Fig. 5a). The addition of both nutrients on day 0 led to the highest amounts of glutamine in the medium with ~ 3.5 mM of glutamine still available in the medium at day 4 with a complete depletion only observed on day 7 or 8. But such combinations of nutrients also exhibited the most deleterious effect from an ammonium production point of view (Fig. 5b). Strategy D appeared the most deleterious with a final concentration of ammonium higher than 7 mM inducing low VCCmax and IVCC (Fig. 4; Table 1). Although strategy E showed a significant increase of VCCmax (6.8 × 106 cells/mL) and IVCC compared to strategy A, it produced greater amounts of ammonium (Figs. 4 and 5b). A similar addition was also tested on day 3 (strategy H) to control the glutamine profile at lower concentration over days and limit the amount of ammonium produced. Although obtaining an intermediate concentration of glutamine in the medium until day 7, no statistically significant difference in both viability and metabolic profiles was observed compared to strategies E and F (Figs. 4 and 5).

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)

To observe the impact of glutamine concentration on cell culture, either 4 mM glutamine or glutamax were added one half on day 0 and the other half on day 3 in strategies I and J, respectively. Glutamine concentration remained between 0.5 and 2 mM until day 7 indicating a lower nutrient consumption by the cells than for strategies A and B (Fig. 5). Considering the metabolic profiles, this limited consumption only slightly decreased the lactate produced, the concentration of the other metabolites remaining comparable to strategies A and B, but such supplementation improved the cell viability and especially IVCC ( ~ + 20% compared to strategy A). The best results were obtained with strategy J that allowed maintaining more than 70% cell viability until day 7 whereas with most strategies it dropped below 70% as early as day 5 (strategies A and D) or 6 (strategies E, H, and I) (see Figures S3 and S4 in Supplementary Information). From these results, we hypothesized that a fed-batch type strategy might be an interesting supplementation strategy.

Several fed-batch-mimicking strategies were thus investigated. The effect of glucose addition was first studied through strategies F and G that corresponded to A and B, respectively with the addition of 3 g/L glucose on days 3 and 6. Although the VCCmax significantly increased (Fig. 4), glucose supplementation during cultivation did not seem to be a good alternative in view of the metabolic profiles observed (see Figure S4 in Supplementary Information). Especially, the lactate concentration sharply increased inducing an acidification of the culture medium. Finally, strategies K and L corresponded to the reference strategy with the addition every 3 days of fresh Optipro SFM medium with or without glutamine, respectively. In strategy M, the initial medium was the glutamax-supplemented one and fresh Optipro SFM with glutamax was added every 3 days. Among these mimicking fed-batch feedings, strategies K and M appeared interesting with VCCmax values similar to those of strategy B (Figs. 4 and 6a) and a viability that remained above 70% until 7 and 8 days compared to 5 and 6 days for strategies A and B, respectively (Fig. 6b). Strategy M exhibited slower growth kinetics with lower µmax and higher tD than the other strategies while no significant evolution of IVCC was observed (Fig. 6a; Table 1). Furthermore, the amounts of ammonium and lactate were among the lowest produced as for strategy B (Fig. 6c). Strategies B and M were thus considered as the most promising strategies.

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)

Scale-up in 3 L-bioreactor and feasibility test

To test the robustness of the DuckCelt®-T17 culture process from shake flask to pilot scale, the cells were cultured in a 3 L bioreactor using the two promising supplementation strategies B and M. Each culture was repeated twice at 100 rpm using 30% dO2 and compared to the reference strategy A.

As observed at lab scale, the substitution of glutamine by glutamax significantly improved the DuckCelt®-T17 cell growth in terms of VCCmax (from 3.4 × 106 cells/mL to 5.3 × 106 cells/mL, p-value ≤ 0.05) (Fig. 7a). The production of lactate remained below 1.5 g/L compared to 2 g/L for the reference strategy A (Fig. 7c), while no difference in ammonium production and viability profile was observed (Fig. 7b and c). The significant improvement of both cell growth and viability using the fed-batch condition (strategy M) was also observed at pilot scale with a VCCmax ~ 5.1 × 106 cells/mL The most promising results in a context of virus production were obtained for the cell viability which remained above 80% during the eight first days of culture against 5 days for strategies A and B (Fig. 7b).

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)

Due to the good results observed with fed-batch cell cultivation, we finally performed a feasibility assay to test the interest of a perfusion strategy. Similar viability profiles were observed with fed-batch and perfusion strategies (Fig. 7b) with a viability maintained above 80% throughout the culture process. But perfusion cell cultivation also greatly improved the cell growth with a VCCmax ~1.1 × 107 cells/mL and limited the concentration of lactate and ammonium in the culture medium below 0.5 g/L and 2 mM at the end of the culture, respectively (Fig. 7a, c and d). On the other hand, this process must be optimized since glutamine and glucose (< 0.5 mM and close to 0 g/L respectively) did not remain available to the cells until the end of the culture in the bioreactor despite their continuous addition (Fig. 7d).