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Purification of Recombinant Vaccinia Virus for Oncolytic and Immunotherapeutic Applications Using Monolithic Column Technology

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Purification of Recombinant Vaccinia Virus for Oncolytic and Immunotherapeutic Applications Using Monolithic Column Technology Purification of Recombinant Vaccinia Virus for Oncolytic and Immunotherapeutic Applications using Monolithic Column Technology A thesis submitted to University College London for the degree of Doctor of Engineering by David Isaac William Vincent The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Gordon Street London WC1H 0AH 1 I, David Vincent confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed……………….………………………Date……………………………………… 2 Acknowledgements I would like to thank my supervisor Dr Yuhong Zhou for her invaluable feedback and support over the course of this EngD. Without her continuous encouragement this work would likely never have been completed. I would also like to thank my Advisor Prof. Ajoy Velayudhan for his help and guidance. Many a long discussion was had on the intricacies of vaccinia viral purification which were thought provoking, inspirational but also really good fun. I would also like to express my gratitude to the staff and students at UCL. There are too many to mention everyone, but in particular I would like to thank Mrs Ludmila Ruban, Miss Dhushy Stanislaus and Dr Tarit Mukhopadhyay for their support, especially in setting up the vaccine lab in the department which I could benefit from in my final year. I would also like to thank Dr Patricia Perez Esteban for her support, friendship and bioprocess wisdom. I would like to express my gratitude to BIA Separations and the EPSRC for sponsoring this work. Without this financial support this work would not have been possible. At BIA Separations I would especially like to thank Dr Aleš Štrancer, Dr Petra Kramberger, and Dr Rosana Hudej for their guidance and technical expertise. At lot of this work was unexpectedly performed at Barts Cancer Institute, Queen Mary University of London. I would like to express my gratitude to the institute for being so accommodating, and especially like to thank Prof Yaohe Wang, Dr Ming Yuan, Dr Rathi Gangeswaren, Dr Chadwan Al-Yaghchi and Dr Jahangir Ahmed for their encouragement, assistance, and friendship. 3 Finally I would like to thank my family for sticking by me during the last 4+ years. I want to particularly thank my amazing wife Ruxandra for her love, support and guidance in all aspects of my life and work. During this work, my beautiful son was born, Joseph John Maurice Comisel-Vincent. Welcome to the world, I hope you find Daddy’s work interesting and useful. Also Angela you owe me £5. 4 Abstract 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 5 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. 6 Impact Statement The gene and immunotherapy field has picked up a lot in the last few years after some initial setbacks in the clinic. A number of in vivo and ex vivo viral constructs have recently entered clinical trials (Heo et al. 2013). Vaccinia virus has shown promising efficacy in both animal models and late stage cancer patients for both solid tumours and models for metastasis (Garber 2006;Kirn and Thorne 2009;Tysome et al. 2009;Tysome et al. 2011). While challenges associated with oncolytic viral therapies do exist (Ferguson et al. 2012), there is a chance that vaccinia virus will need to be produced for direct injection into patients at a commercial scale. The scale of this production will of course depend on the efficacy of the trial, the indication, and the dose required. Clinical trials currently suggest that the total treatment dose requirement will need to be relatively high in order to achieve efficacy. It has been suggested that multiple treatments may be required each consisting of 1x109 pfu per patient (Heo, Reid, Ruo, Breitbach, Rose, Bloomston, Cho, Lim, Chung, Kim, Burke, Lencioni, Hickman, Moon, Lee, Kim, Daneshmand, Dubois, Longpre, Ngo, Rooney, Bell, Rhee, Patt, Hwang, & Kirn 2013). This will not only increase the costs of production but may also result in the requirement for purer material as increasing the amount of viral particles per dose will also increase the amount of impurities per dose, which even at lower doses is already recognised as an issue (Wolff and Reichl 2011). It is clear that without effective, engineering led, process development and optimisation, production costs can become crippling, especially as vaccinia processes to date tend to be low yielding with cell productivity less than 100 pfu/cell (Liu et al. 2016). The impact of this work inside academia will likely be twofold. Firstly, it will advance the levels of process knowledge and understanding of how vaccinia virus Lister strain interacts with charged and hydrophobic monolithic stationary phases. It will also enable 7 laboratories currently working with vaccinia virus Lister strain to produce material of a higher quality, in terms of purity, than previously possible in a single step. This will be especially relevant for groups currently relying on sucrose gradient ultracentrifugation or similar techniques. The wider medical community will indirectly benefit from this as the material supplied for pre-clinical and proof of concept studies will be more representative of that produced in late stage clinical and commercial processes. From an industrial perspective, the impact of this work, at least in the short term, will be the addition of another purification tool in the vaccinia virus purification development toolbox. The reason why this is important is that vaccinia is a challenging virus to purify due to its large size and complexity, and therefore the number of purification options available is limited. The default unit operation used to purify vaccinia and other viruses is still ultracentrifugation. This work will add to the growing debate on whether this technology is still of value. It adds insight into the associated cost of goods for both technologies, suggests potential mechanisms involved in the separation of vaccinia using CIM monoliths, and presents encouraging results indicating the production of highly pure vaccinia virus material using chromatography. 8 Table of Contents Acknowledgements ................................................................................................................... 3 Abstract ....................................................................................................................................
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