The project focused on the Ultra Scale-Down (USD) modelling of harvest and early purification for viral vaccine and vector production processes. USD models seek to mimic the commercial scale process environment using only millilitres of material. Many commercial processes include a disc-stack centrifuge, micro-filtration, tangential flow ultrafiltration and density gradient ultracentrifugation steps, which are investigated in this work. A key parameter of USD, in particular for centrifugation, has been shown to be shear, which may cause significant damage to biological products and increase the difficulty of subsequent processing steps. To quantify shear rate therefore the PSC-5 disk-stack centrifuge and PKII density gradient ultra-centrifuge have been modelled using Comsol Multiphysics®. This EngD project extended existing Computational Fluid Dynamic (CFD) analysis by treating the centrifuge as a 3 dimensional system, drawing comparison to the rotating disc shear device based on a simple population balance analysis (accounting for different shear rate levels and time of exposure), and identifying the nature of the shear (simple versus extensional). This modified approach produced a good prediction of shear rate as validated against experimental results, as well as intuitively representing a closer match to the physically important points of comparison between the two systems. In order to overcoming the challenge of experimentally validating CFD predictions in downstream process units a novel approach was adopted: use of synthetic particles that mimic mammalian cells. Adju-Phos was applied successfully for the prediction of shear rate in a number of pilot scale centrifuges of both tubular bowl and diskstack design, by correlating changes in the particle size distribution against that seen in the USD shear device. The significant advantages of using Adju-Phos include reduced cost, pre-work and time. For the PKII density gradient ultracentrifuge, a smaller virus shear mimic, consisting of an off-the-shelf polystyrene nanobead coated with streptavidin, was used for experimental validation of CFD. This work also represents the first steps in developing an USD model for the continuous feed industrial ultracentrifuge, consisting of the rotating disc shear device and laboratory ultracentrifuge matched to the large scale on the basis or 2 t and head at the product layer. The fact that these nanobeads showed an impact from hydrodynamic forces despite their small size, raised the possibility that viruses, which had been thought to be too small for damage by hydrodynamic or other forms of shear, might also be susceptible. There is limited if any literature on the impact of shear rate on cells used to produce viral products, or on the viral products themselves. The existing USD tool was used to provide some insight into the performance of the harvest step for influenza and adenovirus production processes in continuous cell-lines. The results indicated that virus infected cells do not actually show any increase in sensitivity to shear forces, and may indeed become less shear sensitive, in a similar manner to that previously observed in old or dead cell cultures. Clarification may be most significantly dependent on the virus release mechanism, with the budding influenza virus producing a much greater decrease in clarification than the lytic, nonenveloped adenovirus. A good match was also demonstrated to the industrial scale performance in terms of clarification, protein release and impurity profile. The impact on the influenza and adenoviruses themselves was measured using infectivity assays, TEM and particle size distribution and protein contents of post shear purified viral particles. Whilst no impact was recorded based on the infectivity assays or TEM, a small but significant change in particle size distribution was shown for adenovirus, and is indicated for influenza. The influenza virus also showed a possible change in protein make-up of purified viral particles, indicating possibly a loss of those proteins from the virus structure. The significance of these morphological changes, if any, remains to be confirmed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:747692 |
| Date | January 2018 |
| Creators | Melinek, Beatrice |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/10048337/ |
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