An engineering characterisation of shaken bioreactors: flow, mixing and suspension dynamics

The thesis describes an experimental investigation of the flow, mixing and suspension dynamics in cylindrical orbitally shaken bioreactors (OSRs). Amongst the plethora of bioreactor types and geometries available for cell culture, the OSR is ubiquitous in bioprocess research and development. Offering a well defined liquid-gas interface, high throughput potential and experimental flexibility, it is the vessel of choice in early bioprocess research, either as microtiter-plates, Erlenmeyer flasks or other geometries. Despite recent advances in the field, an accurate and exhaustive engineering characterisation of OSRs from this point of view is lacking. In the present study a mixing time estimation methodology is developed and employed to assess the effect of operational parameters on the mixing of cylindrical OSRs. Particle Image Velocimetry measurements are carried out to evaluate the effect of vessel geometry modifications on the flow. Laser Induced Fluorescence is used to produce accurate description of the micro-mixing. Solid suspension studies are also undertaken to assess potential strategies to improve microcarrier culture in OSRs. Accurate determination of the mixing time in OSRs is essential for the optimisation of mixing processes and minimization of concentration gradients that can be deleterious to cell cultures. The Dual Indicator System for Mixing Time (DISMT) is employed, together with a purposely built image processing code to objectively measure mixing times in cylindrical and Erlenmeyer flask bioreactors. Relevant data acquisition aspects to optimise the accuracy of DISMT measurements are discussed in detail, with direct comparison of different mixing time measurement methodologies, including DISMT, pH probe and iodine thiosulfate decolourisation results obtained in two types of stirred reactors. The DISMT is employed to determine mixing characteristics of OSRs at different flow conditions and develop an effective feeding strategy, by evaluating the effect of the position of the feed at different radial locations in the vessel. At low Fr the flow presents a toroidal vortex below the free surface, which controls mass transport process across the entire vessel and defines two distinct regions in and outside of the vortex exhibiting different mixing rate. At higher Fr the axial flow enhances the mixing of the fluid located next to the wall as the mean flow transition to axial flow coincides with a regime flow transition and onset of turbulence fluctuations. By controlling the locations of eed addition, the flow characteristics can be exploited to enhance initial distribution of the added liquid and decrease the time required to reach homogeneity. The mixing number is highly dependent on the position of the feeding pipe. Insertion close to the vessel walls, and in the periphery of the toroidal vortex, where local shear stresses and deformation rates are highest, were found to significantly enhance mixing. In order to provide an effective scaling methodology, the results obtained in OSRs are compared with data previously reported in the literature for both cylindrical reactors and Erlenmeyer flasks. The employment of a critical Froude number shows promise for the establishment of a scaling law for mixing time across various types and sizes of shaken bioreactors. The flow dynamics in cylindrical shaken bioreactors of different conical bottom geometries (inward facing) is investigated by means of phase-resolved Particle Image Velocimetry. The cylindrical bioreactor with a conical bottom geometry is selected to assess its potential application in three-dimensional cultures, and improve solid suspension in shaken systems. The effects of conical shaped bottoms of different heights on the fluid flow are evaluated for different operating conditions with water being the working fluid. The results provide evidence that the presence of the conical bottom affects the transition from laminar to turbulent flow, increases the vorticity and generates shear stresses at well defined locations. The increased kinetic energy content measured with PIV in cylindrical OSRs with a conical bottom is found to effectively enhance solid suspension in microcarrier or embryod body cell culture. The dynamics of solid suspension is studied using commercially available Cytodex-3, stained with trypan blue for improved visual contrast and image acquisition. The presence of the conical bottom improves solid suspension by requiring lower agitation rates for the microcarriers to lift from the bottom completely. The critical Froude, which determines the flow type controlling the bioreactor, can also be used to scale the suspension of microcarriers in OSRs. Full characterisation of macro- and micro- mixing scales in OSRs for highly viscous is obtained by DIMST and pLIF, respectively. This data also provides an effective visualisation of the flow structures controlling the bioreactor transport processes. The mixing characteristics of high viscosity fluids in OSRs are investigated by means of DISMT and pLIF, for the macro- and microscales of mixing, respectively. Fluids of viscosities 2-14 times that of water, exhibit flow characteristics different to those observed at ν=10−6 m^2s^−1. A toroidal vortex, similar to that observed in water, is present at low Fr. Small increments of agitation rate provide transition to other flow structures, never reported in the literature. The pLIF measurements allow to characterise the small scale features of the flow not observable from phase averaged PIV, and visualise well defined elongation and striation dynamics for different regimes. Although extensively used, OSRs are yet to be fully characterised. Further research is required on the hydrodynamic phenomena dominating orbitally shaking vessels, to enable the development of scale-up platform to simplify and speed progress from cell line development to industrial production of bio-products. The development of the scaleup platform must be made considering the advantages and requirements of single-use technology, to provide the industry with robust and reproducible scale-up model.