This study involved the evaluation and optimisation of a 4 x 500 ml MBR system that is capable of parallel mammalian cell culture. This unit also has the capability of operating 16 parallel bioreactors hence providing the potential for an effective, high throughput process development/optimisation tool. The MBR system was mechanically and geometrically similar to a standard 5L STR that was used to carry out scale comparison studies.
Initial design modifications were made to the hardware to make it suitable for mammalian cell culture. This included designing and fitting a sampling mechanism that provided a simple to use solution. Also a variety of sparger designs were produced to replace the 15 µm sintered sparger that was provided with the unit due to its excessive foaming in CD-CHO media.
A comprehensive engineering characterisation of the MBR and 5L STR was conducted to evaluate the MBR system’s performance and in order to facilitate scale comparison studies. Characterisation included mechanical power input, kLa, mixing time and hydrodynamics. Power input was measured for the directly and magnetically stirred MBRs by using a small scale air bearing dyanometer. The Np was calculated for both agitation configurations with the Np for the direct driven impeller equalling 0.42. The Np for the magnetically driven impeller was not accurately measured due to limitations with the experimental set up, however at higher impeller speeds the Np for both impeller types became similar. The Np
of the 5L STR was measured previously as 0.97 (Nick Silk, personal communication). The 5L STR was run at 260 rpm and at this speed it had an estimated P/V of 20.5 W/m3. At matching P/V the agitation speed of the directly driven MBR was estimated to be 410 rpm and 350 rpm for the magnetically driven MBR.
kLa was measured for the MBR system and the 5L STR as a function of agitation rate using the static gassing out method with CD-CHO media at 37°C, as this mimicked cell culture conditions. kLa measurements were highest for the 90 µm
sintered sparger design for both the directly and magnetically driven impellers (16-24 h-1). On average the kLa measurements obtained for the other sparger designs were similar and varied between 5-14 h-1. kLa was also measured for the MBR using surface aeration and the values obtained were between 1.25-2.25 h-1. The kLa for the 5L STR was measured at different agitation and gas flow rates (100-260 rpm and 0.03-0.06 vvm). The kLa at 0.03 vvm and P/V = 20.5 W/m3 was 3.8 h-1, doubling the gas flow rate only increased the kLa to 4.6 h-1. It was found that the agitation rate had a more profound effect on kLa than gas flow rate which is consistent with literature data for bench and pilot scale reactors (Van’t Riet, 1979).
Mixing times were measured for the MBR system using both a decolourisation method and the pH tracer method as a function of both agitation rate and fill volume. Mixing times reduced significantly at faster agitation rates and with smaller fill volumes. The directly driven impeller produced lower mixing times compared to the magnetically driven impeller. The 5L STR had a mixing time of 6 seconds at P/V = 20.5 W/m3 which was very similar to the directly driven MBR which produced a mixing time of 6.3 seconds at matched P/V. It was found that the mixing times measured using the decolourisation method with the directly driven impeller were similar to those predicted by a correlation developed by Nienow (1998).
Tm = 5.9DT2/3
( Ɛ T)-1/3(Di/DT)-1/3 6.1
The hydrodynamic conditions in the MBR and 5L STR were evaluated and their potential impact on the cells was analysed. It was found that at a matched P/V of 20.5 W/m3 the Kolmogorov eddy sizes in the impeller region of the directly and magnetically driven MBRs and the 5L STR was above the average cell diameter.
Hence it was found that the conditions produced by the reactor systems at matched P/V should not have resulted in substantial mechanic shear damage on the cells.
An initial batch scale comparison cultivation was conducted comparing the performance of the directly and magnetically driven MBRs and the 5L STR.
Constant P/V was used as a scale down factor 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 specific productivities obtained in the magnetic and directly driven MBRs were higher than that achieved in the 5L STR (26 pg/cell/day in the magnetically driven MBR; 21 pg/cell/day in the directly driven MBR and 16 pg/cell/day in the 5L STR).
A series of batch cultivations were carried out in the MBR system using the directly driven impeller to optimise its performance. These cultivations focused on optimising the gas delivery mechanism to the system and evaluated surface aeration and gas sparging. These cultivations showed that the design and operation of different gas delivery configurations can have profound effects on cell culture performance. The 90 μm sintered sparger was found to be unsuitable for use with the GS CHO cell line trialled in these cultivations. It was shown to significantly hinder cell growth to the extent that the cell culture did not enter into the exponential growth phase. The configuration that involved gas sparging using the singular opening 0.31 cm diameter 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. The configuration that involved surface aeration produced 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. 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.
Scale comparison cultivations were carried out with the MBR and 5L STR at a matched P/V of 20.5 W/m3 and matched mixing time of 6 seconds. Gas was delivered to the culture via surface aeration as this mode of gas delivery resulted in better cell growth compared to direct gas sparging. This scale comparison showed that the directly driven MBR using surface aeration can successfully
perform high cell density fed-batch cultivations. The growth profiles and the antibody productivity for the MBR and 5L STR showed good similarity (refer to Section 5.2.1).
It was also shown that the MBR system can be adapted to enable continuous feeding using extremely low flow rates of between 0.1-0.2 ml/hour. The MBR was capable of maintaining fairly tight control around the culture target glucose concentration of 2.0 g/L after feed initiation, with the lowest concentration being 1.69 g/L and the highest being 2.26 g/L. This means that culture glucose concentration was kept within 2.0 ± 15 % g/L.
In summary, the BioXplorerTM MBR system is capable of producing similar growth kinetics and product titres to lab-scale bioreactors and hence offers an economical and user friendly scale-down process development/optimisation technology. Its working volume of ~ 300 ml provides enough culture volume to facilitate regular sampling for offline analysis. It is also capable of performing bolus and continuous feeding operations both of which are now standard and essential elements of modern industrial mammalian cell culture processes.