Optimisation of virus replication in spinner flasks
To optimise measles virus production in MRC-5 cells, the effect of regulating the glucose level at 1 g/l, culture medium and the MOI, were investigated.
Cells were first grown in MEM + 5%FCS, on 3 g/l Cytodex 1. Once the cell density reached the highest level (around 1.5 to 2 × 106 cells/ml), cells were washed twice with the medium to be tested: M199, M199 + 2% FCS, M199 + 0.2% gelatin and M199 + 0.5% BSA (Bovine serum albumin). Cells were infected at day 7, at an MOI of 0.01 with AIK-C measles virus strain.
After cell infection, we observed a continuous decrease of cell density, concomitantly virus titer increased. The maximal virus titer was reached 4 to 5 days after cell infection and was equal to 106.5, 106.75, 106.62 and 107 TCID50/ml, in M199, M199 + 2% FCS, M199 + 0.2% gelatin and M199 + 0.5% BSA, respectively (Fig. 1).
Based on these data, it appears that the enrichment of M199 medium with various components did not result in a dramatic increase of virus titer, and comparable yields were observed for all conditions. Therefore, M199 medium was considered as an acceptable medium for measles virus replication in MRC-5 cells and was chosen for further optimization studies.
The effect of regulation of glucose level at 1 g/l during cell infection phase is illustrated in Table 2. We achieved a maximal virus titer of 106.75 and 106.875 TCDI50/ml, for the regulated and non-regulated glucose level conditions, respectively. The specific productivity was equal to 0.64 and 1.14 infectious virus cell−1 day−1. Hence, it appears that maintaining glucose concentration at 1 g/l during cell infection phase did not enhance virus replication.
To study the effect of MOI on measles virus productivity in MRC-5 cells, three levels (0.01, 0.001 and 0.005) were tested. Cells were first grown in MEM + 5% FCS on Cytodex 1 at 3 g/l, when the maximal cell density was achieved (around day 5); cells were washed twice with M199 and infected at the required MOI.
Data shown in Fig. 2 show a typical behaviour of cells upon their infection, i.e., a continuous decrease of cell density with a gradual increase of virus titer. The highest virus titer was obtained at an MOI = 0.01, 7 days post infection and was equal to 106.875 TCID50/ml. The use of an MOI of 0.005 resulted in a maximal virus titer of 106.25 TCID50/ml. For the lowest MOI (0.001), the maximal virus titer reached 105.875TCID50/ml. Therefore, it appears that cell infection at an MOI of 0.005 can be used without a significant loss of cell productivity. Hence, this MOI was selected for bioreactor culture optimization.
Process optimization in a 7-l bioreactor
To develop the process of measles virus production in MRC-5 cells grown on Cytodex 1 microcarriers in a stirred bioreactor, we first optimized cell growth phase then the virus replication phase.
Cell growth phase
To optimize MRC-5 cells growth on Cytodex 1 microcarriers in a 7-l bioreactor, we first studied the effect of culture medium. MEM was used as a basal medium; in addition, MEM + 5% FCS diluted with PBS to 75% and 50% were assessed for their ability to sustain MRC-5 cell growth. The initial glucose and glutamine levels were equal to 3 g/l and 4 mM, respectively.
Data depicted in Table 3 show that the highest cell density level was reached when non-diluted MEM + 5% FCS was used as a growth medium. Under these conditions, the cell density level was equal to 5.2 × 106 cells/ml. We achieved 4.1 × 106 and 3.4 × 106 cells/ml, when MEM + 5% FCS medium was diluted with 25% and 50% PBS, respectively. However, the highest average specific growth was reached in the 25% PBS-diluted medium. The average specific growth rate (μ
avg) observed in the non-diluted medium was slightly lower (0.03 versus 0.034 h−1). In addition, data shown in Table 3 indicate a similar level of the specific medium consumption rate for the 25% PBS-diluted and the non-diluted media. For the 50% PBS-diluted medium, the specific medium consumption rate was slightly lower in comparison to the other conditions. Nevertheless, in this case cell density level was 0.8-fold lower than that observed in 25% PBS-diluted medium. Metabolites levels were comparable for all the media, although the lowest lactate level was obtained in the 25% PBS-diluted medium. Regarding glutamine profile, a limitation was observed on day 5 of the culture, for the 50% PBS-diluted medium (data not shown).
The analysis of these data shows that 25% PBS-diluted medium enables to achieve a high cell density level with an efficient use of the medium. This medium was therefore selected for subsequent optimization studies.
Virus replication phase
To optimize the virus production phase in MRC-5 cells grown on 3 g/l Cytodex 1 microcarriers, the effect of the culture mode and perfusion rate were investigated.
Various cultures were conducted using either perfusion or repeated batch as a culture mode during the virus production phase. Cell growth step was always conducted in recirculation in 25% PBS-diluted MEM + 5% FCS.
Figure 3a indicates that the highest cell density was obtained at day 7, and was equal to 4.3 × 106 cells/ml. At this moment, cells were infected at an MOI of 0.005; the culture was then conducted using either perfusion or repeated-batch culture mode. After cell infection, a continuous drop of cell density was observed, while we observed a progressive increase of the virus titer, for both cultures. The highest titer was equal to 107 TCID50/ml and 7.5 × 106 TCID50/ml, for the perfusion and the repeated-batch cultures, respectively. Nevertheless, this level was reached at different times post infection.
Nine harvests were collected when perfusion was used during the virus replication phase. For the repeated-batch culture, only two harvests were obtained. The specific productivity was equal to 1.6 and 0.03 infectious virus particle [IVP] cell−1 day−1 for the perfused and repeated-batch cultures, respectively. Therefore, using perfusion during the virus replication phase resulted in a higher virus yield when compared to repeated batch. Thus, perfusion was selected as the optimal culture mode for the virus production step.
Figure 3b indicates that glucose level was not limiting during perfused and repeated-batch cultures. Lactate levels observed during cell growth phase were comparable for both cultures. Nevertheless, a higher level of lactate was noticed by the end of the perfused culture. At day 14, lactate levels were equal to 13.5 and 10 mM for the perfused and repeated-batch cultures, respectively.
Regarding glutamine and ammonia levels, no glutamine limitation was observed for both cultures. Average ammonia level was around 3 mM during the cell growth phase. However, residual glutamine level decreased significantly after cell infection when the culture was conducted either in perfusion or repeated-batch culture mode. During this phase, a lower amount of ammonia was produced by the cells, for both cultures.
In an attempt to increase the productivity, we carried out a culture where a lower perfusion rate (0.1 V/day) was applied during the virus production step (Fig. 4). Comparable levels of cell density were obtained during both cultures (Fig. 4a). In addition, similar average specific growth rate, around 0.026 h−1, was observed. Cells were infected at day 7, at an MOI of 0.005, as previously described. After cell infection, the culture was conducted in perfusion at a rate of 0.1 V/day.
Time course of virus titer shows that we obtained a slightly lower titer when compared to the culture perfused at 0.25 V/day. Nevertheless, in this case the virus replication phase was 1 day longer than the 0.25 V/day culture, resulting in a further harvest.
However, the comparison of the specific productivity indicates that the highest specific productivity was reached for the 0.25 V/day culture; its level was 148-fold higher when compared to the 0.1 V/day perfused culture. We achieved a specific productivity of 1.6 IVP cell−1 day−1 for the highest perfusion rate culture.
Regarding substrate (glucose and glutamine) and metabolite (lactate and ammonia) profiles, glucose level was not limiting during the whole duration of the culture, for both conditions (Fig. 4b). An increase of lactate was observed as glucose was consumed. Similar profiles for glutamine and ammonia are shown in Fig. 4c, although a drastic decrease of glutamine level was observed during the virus production step, as previously shown in Fig. 3c.
Stability data of the vaccine using various stabilizers are shown in Fig. 5. Residual infective virus was highly dependent on the stabilizer used and the storage temperature. The storage stability was the best for the S1-stabilized vaccine. This vaccine exhibited the lowest loss in infectious virus titer under all conditions. At room temperature (around 28 °C), the stability of this vaccine was remarkably higher when compared to the non-stabilized and the S2-stabilized vaccines (Fig. 5). No infectious virus was detected after 3 and 6 days of storage at room temperature for the S2-stabilized and the non-stabilized vaccines, respectively, whereas the S1-stabilized vaccine retains its infectivity to a significant extent after 7 days of storage.
Similar findings were observed at +4 °C and −60 °C temperatures. Nevertheless, the S2-stabilized and the non-stabilized vaccines were more stable under these conditions as compared to the storage at room temperature.
All vaccines showed the highest stability when stored at −60 °C. At this storage temperature, the loss in virus titer was the lowest for all vaccines.