Vaccinia virus Lister strain, a large, structurally complex double stranded DNA pox virus, is being developed by a number of organisations around the world as an oncolytic and immunotherapeutic agent for the treatment of a broad range of cancers. Should these therapies prove to be efficacious in the clinic, large quantities of vaccinia virus will need to be produced at very high levels of purity as dosing requirements are expected to be as high as approximately 1x109 pfu/dose. In this thesis, the development of two convective interaction media (CIM) monolith capture steps for vaccinia virus with considerable purification factors is described; one uses cation exchange while the other uses hydrophobic interaction chromatography. The purification process development involved an extensive material characterisation study resulting in enhanced product understanding, a rapid resin screening study aimed at quickly identifying suitable resin chemistries, followed by process optimisation studies on the best performing monoliths. After being challenged with crude infectious vaccinia harvest, CIM OH monoliths are shown to be able to recover up to 90% of the infectious virus loaded whilst removing up to 99% of the contaminating DNA (without nuclease treatment) and 100% of quantifiable protein. Binding capacities were shown to be in the order of 1x109 pfu/mL. The high levels of both batch to batch and assay variability as well as the tendency of vaccinia virus to aggregate in the feed material, typical of viral processes especially when developed alongside un-optimised upstream conditions, are clearly demonstrated and the implications are explored. The results show that it can be challenging to draw robust conclusions on process performance. To minimise the effects of analytical variability, a number of orthogonal analytical methods have been used to quantify and characterise viral particles. These include TCID50, nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and real-time PCR (qPCR). In order to put this into an industrial context, a comparative cost of goods analysis of monoliths and ultracentrifugation technologies for the purification of large viral particles is provided. This shows that both chromatography using monoliths and continuous flow ultracentrifugation can be economically viable, although both have limitations. The potential economic benefits of using a monolith-based process over an ultracentrifugation-based process are increased productivity, the ability to generate purer material and ease of scale-up. CIM monoliths are unique stationary phases that offer efficient separation and high productivity owing to fast cycle times and high binding capacities. Both cation exchange (CIM SO3) and hydrophobic interaction (CIM OH) monoliths are effectual at removing the majority of contaminants in a single purification step that can easily be scaled up to 8 L bed volumes. CIM monoliths have the potential to be an attractive option for future manufacturing processes for oncolytic viral therapies. They are shown in this thesis to achieve a higher percentage recovery and better removal of DNA, protein and aggregate than any other technology described in the literature to date.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:747038 |
| Date | January 2017 |
| Creators | Vincent, David Isaac William |
| 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/10038211/ |
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