Chapter 4: Optimisation of gas delivery to the MBR system
4.8 Evaluation of the effects of antifoam on cell growth
To eliminate the possibility that antifoam added to the cultures employing direct sparging may have contributed to the reduced growth in these cultures, a shake flask experiment was conducted to evaluate the effects that antifoam can have on growth. One flask was run as a control with no antifoam addition. The experimental flask received 0.004 v/v of 3% antifoam solution every day from day 2 onwards which is slightly more than that which would have been added to the MBR using the 90 μm sintered sparger. This experiment utilised 1 L shake flasks with a culture volume of 300 ml to best mimic the culture volume in the MBR.
Figure 4.22: Growth profiles for batch flask cultivations run at 37°C, 140 rpm and 5% CO2. VCC for control flask 1 with no antifoam (♦); VCC for flask with 0.004 v/v of 3% antifoam added daily from day 2 (■); cell culture viability for control flask (◊) and cell culture viability for flask with antifoam added (□).
The growth curves in figure 4.22 are almost identical which would indicate that the quantity and concentration of antifoam added to the cultures did not have any effect on growth. Culture viability for both flasks remains above 95% at the end of the exponential phase at around 192 hours.
0
Figure 4.23: Cell diameter measurement profiles of the control and experimental batch flask cultivations. Control flask (♦) and experimental flask (
■
). Cell size was measured using the Vi-CELL as described in Section 2.7.The cell size profiles are also similar for both the experimental conditions which would also indicate that there was no effect on cell physiology (refer to Fig.
4.23). A metabolite analysis was undertaken at 192 hours (day 7) near the end of the evaluation (refer to Table 4.3). The final metabolite concentration and osmolality of both culture conditions were very similar again indicating that the difference in culture conditions did not have a significant effect.
This evaluation indicates that the quantities of antifoam added to the MBR would not have contributed to poorer growth as the same concentration of antifoam added to shake flask cultures did not affect growth.
14.0 14.5 15.0 15.5 16.0 16.5
0 24 48 72 96 120 144 168 192 216
Cell diameter (μm)
Time (hours)
318.97.81140.0042.3801.671.7Concentration
Osm (mOsm/Kg)K+ (mmol/L)Na+ (mmol/L)Glu (mmol/L)NH4+ (mmol/L)Gln (mmol/L)lac(g/L)Gluc (g/L)Metabolite
Flask 2
322.97.31170.332.6301.471.89Concentration
Osm (mOsm/Kg)K+ (mmol/L)Na+ (mmol/L)Glu (mmol/L)NH4+ (mmol/L)Gln (mmol/L)lac(g/L)Gluc (g/L)Metabolite
Flask 1 318.97.81140.0042.3801.671.7Concentration
Osm (mOsm/Kg)K+ (mmol/L)Na+ (mmol/L)Glu (mmol/L)NH4+ (mmol/L)Gln (mmol/L)lac(g/L)Gluc (g/L)Metabolite
Flask 2
322.97.31170.332.6301.471.89Concentration
Osm (mOsm/Kg)K+ (mmol/L)Na+ (mmol/L)Glu (mmol/L)NH4+ (mmol/L)Gln (mmol/L)lac(g/L)Gluc (g/L)Metabolite
Flask 1
Table 4.3: Metabolite concentrations and osmolality of the control (flask 1) and the flask with added antifoam (flask 2). Samples were taken at 192 hours.
4.9 Conclusions
These experiments have shown that the design and operation of different gas delivery configurations can have profound effects on cell culture performance.
An initial scale comparison was conducted comparing the performance of the directly and magnetically driven MBRs and a 5L STR. Constant P/V was used as a scale-down criterion which also resulted in a constant mixing time for the 5L STR and the directly driven MBR. There was a significant difference between the maximum VCC of the 5L STR (9.9 x 106 cells/ml) and both the magnetic and direct driven MBRs (5.1 and 4.9 x 106 cells/ml respectively).
The Qpof the magnetically and directly driven MBRs were twice that of the 5L STR (24 vs 12 pg/cell/day) when plotting all of the product concentration data points against IVCC. Qp values differed however, when lines of best fit were plotted resulting in a Qp of 26 pg/cell/day for the magnetically driven MBR, 21 pg/cell/day for the directly driven MBR and 16 pg/cell/day for the 5L STR. It must be noted that these Qp figures are estimates and may have a certain degree of error due to variability in the product concentration data.
It was found that the 90 μm sintered sparger is not suitable for use with the cell line trialled in these experiments. Compared to the sintered sparger, the configuration that involved sparging directly into the culture using the singular opening 0.31 cm sparger design yielded far better culture performance. It produced a maximum VCC of 6.8 x 106 cells/mL, a μmax of 0.023 h-1 and a product titre of 0.58 g/L. Furthermore, the configuration that involved surface aeration produced even better cell growth reaching a maximum VCC of 8.1 x 106 cells/mL and a μmax of 0.028 h-1, however it produced a slightly lower titre of 0.52 g/L. Interestingly, the culture that involved direct sparging produced cells that had a significantly higher cell specific productivity of 16 pg/cell/day compared to 10 pg/cell/day in the cultivations that employed surface aeration.
The main results are summarised in table 4.4.
Table 4.4: Summary of the main performance parameters for each gas delivery configuration. These figures are averages based on duplicate MBR runs. The numbers in the brackets are ± 1 standard deviation about the mean.
Configuration Maximum VCC
(x106 cells/mL) μmax (h-1) Titre (g/L) Qp (pg/cell/day)
90 μm sintered 1.34 NA NA NA
Direct sparging
- 0.31 cm 6.8 (± 0.36) 0.023 (± 0.0011) 0.58 (± 0.0057) 16 (± 1.7) Surface aeration 8.1 (± 0.22) 0.028 (± 0.0022) 0.52 (± 0.042) 10 (± 0.92)
The difference in performance between the cultivations operated with the direct gas sparging method (singular opening 0.31 cm sparger) and cultivations operated with surface aeration could be due to the effects of the culture being drawn up the sparger shaft in between sparging. This phenomenon could have stressed the cells by removing them from the controlled environment within the bulk culture into an uncontrolled and potentially hostile environment within the sparger shaft. The cells may also have been impacted by shear forces when being discharged out of the sparger shaft back into the culture due to shear forces created by wall of the sparger shaft.
The work in this chapter has shown that optimising the gas delivery system of the directly driven MBR improved cell growth. These cultivations, however, do not provide a direct comparison with the 5L STR because the optimisation cultivations were run in batch mode while the initial cultivations (Section 4.2) were run in fed-batch mode. In order to progress the scale-down comparison work further; a direct comparison between the directly driven MBR system and the 5L STR run in fed-batch mode at a matched P/V of 20.5 W/m3 is therefore required.
As surface aeration in the MBR produced better growth compared to the direct sparging configuration; this mode of gas sparging is used in the next comparison cultivation. Although operation of surface aeration produces a different hydrodynamic environment in the MBR to that produced by direct sparging in
the 5L STR; using surface aeration in a scale-down comparison will evaluate if the MBR system has the potential to match the performance of the 5L STR and sustain high cell density cultures. If performance is found to be comparable, then future work should focus on optimising the MBR to achieve comparable scale-down cultivations using direct sparging.