As an avian cell line, DuckCelt®-T17 VCCmax remains in the low range when compared to the maximum densities reached by AGE1.CR (4–20 x 106 cells/mL) [13] or EB66 cells (8–30 x 106 cells/mL) [10]. Their growth performance, although satisfactory, was slightly lower than some mammalian cell lines classically used for virus production such as Vero (1.0 x 107 cells/mL) [24], MDCK (1.3 x 106 cells/mL) [4] and PER.C6 cells (1 x 107 cells/mL) [6]. After 5 days, their viability loss was confirmed by their metabolic kinetic profiles, consistent with the literature. It is generally agreed that lactate and ammonium should not exceed 1.8 g/L and 2 mM, respectively in the medium to minimize their impact on growth [25] and that the ammonium detrimental effects are often reached at concentrations approximately ten-fold less (e.g., 2–4 mM) than lactate (e.g., 20–40 mM, i.e. 1.8–3.6 g/L) [26]. Thus, decreasing lactate at the expense of even a small increase in ammonium is certainly not desirable.
Oxygen is an essential parameter for aerobic cells’ growth and metabolism [27] and in a bioreactor, dO2 is the classical parameter used to control the oxygen supply to the cells. The challenge is to meet the oxygen requirements of the cells without causing cell damage due to bubble burst [28]. Oxygen requirements vary depending on the cell type and the cell line. For example, CHO cells require high levels of oxygen to be very efficient (VCCmax around 2.107 cells/mL) and hence dO2 is regulated between 30 and 60% with pure oxygen [29, 30]. Avian cells such as the AGE1.CR line are usually grown at 40–55% dO2 by pulsed aeration with air enriched with 7.5% CO2 and 20% O2 [13, 14] or by O2 sparging [18]. For the EB66 line, the dO2 is set at 50% [10, 11] and for CAP cells, it is around 40–50% by pulsed aeration with pure oxygen [31]. Between 10 and 50% dO2, the DuckCelt®-T17 line had similar VCCmax, but its viability dropped at 50% dO2, possibly due to the more frequent air supply resulting in a greater hydrodynamic stress and cell damage. Such issues could be addressed using pure oxygen or other sparger types but our configuration did not allow the use of oxygen. The slow cell growth observed at 10% dO2 was also associated with better viability, which could be interesting during a virus production phase. But it could be too risky for subsequent virus production if the exponential phase of growth (µmax and tD) were not efficient enough to support an infection and if oxygen limitation occurred. In addition, the overproduction of metabolic waste products observed at 10% dO2 may have a detrimental influence on viral production performance over time. From an ammonium and lactate production point of view, the intermediate O2 supply condition at 30% dO2 appeared as a good compromise since ammonium was produced in greatest amounts at 50% dO2 and lactate concentrations were well above 1.8 g/L at 10% dO2 at the end of the culture.
It is well known that the amounts of nutrients in the medium, essentially glucose and glutamine, are also a limiting criterion for cell growth [32], their rapid depletion leading to a drop in cell viability. In our reference experiment, the glutamine and glucose depletion at day 5 could explain the decline in cell growth. Consequently, we decided to down-scale the culture process using shake flasks, one of the best alternative for carrying out experiments at small-scale level because of their easy operation and lower cost [33]. Due to glutaminolysis and glycolysis associated with the Krebs cycle, the consumption of glucose and glutamine is associated with a high production of lactate and ammonium [25]. Many strategies such as the replacement of glucose with alternative sugars, adapting cells to a lactate-supplemented medium [34], using fed-batch processes [32, 35], were developed to reduce lactate or ammonium accumulation. Moreover, substitution of glutamine with other nutrients such as glutamate is one of the strategies used to reduce the amount of ammonium produced by the cells [36].
To improve the cell growth, we especially investigated the effect of glutamax, since this 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. In shake flasks, glutamax allowed increasing growth yields without improving cell viability or metabolic profiles except for lactate production. The same improvements were observed by mimicking a fed-batch process by adding glutamine- or glutamax-supplemented fresh culture medium every three days. Additionally, cell viability was kept above 70% over 7–8 days. When scaling-up to a 3L bioreactor, the same results were obtained with the fed-batch strategies. The fed-batch condition seems the most promising in view of a virus production.
Overall, DuckCelt®-T17 cells are very sensitive to substrate limitation and inhibited by the waste products, ideally requiring the continuous renewal of the culture medium for both continuous nutriment supply and waste products removal. That is why we performed a feasibility perfusion test, this technique allowing a continuous addition of fresh culture medium across fiber membranes that retain the cells while removing waste products and spent medium. The cell retention is achieved by alternating tangential flow filtration which appears as an process especially adapted to virus production such as influenza A [38]. To our knowledge, this is the first time that such satisfying performances were obtained at this scale with the DuckCelt®-T17 cell line. This is of high interest in a context of virus production.
The DuckCelt®-T17 culture process was successfully scaled-up from shake flasks to a 3L bioreactor using different supplementation strategies but with a lower culture performance in the latter. These results could be explained by the aeration and hydrodynamic conditions that are not well-controlled in shake flask systems making them difficult to scale-up [28, 33]. The stirring rate as well as the aeration conditions used at pilot scales induce shear stresses that may cause cell damage [39]. Further investigations are required for scale-up purpose especially to deeper characterize the state of the cells and their O2 consumption during the culture process and as a function of the operating conditions.