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Impact of Engineering Flow on the Release and Recovery of Disabled HSV from BHK Cells

Lotfian, Pantea; (2004) Impact of Engineering Flow on the Release and Recovery of Disabled HSV from BHK Cells. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

With the growing interest in therapeutic genes for the treatment of many life-threatening and life-limiting diseases, the need for highly purified material in large amounts has become urgent. In this thesis the focus is on identifying major bottlenecks in the current general laboratory methods used in the downstream recovery of disabled Herpes Simplex Virus (HSV) propagated in Baby Hamster Kidney (BHK) cells in culture, and to define process solutions to overcome them. After analysing the laboratory scale viral production, two major bottlenecks were identified and assessed in this thesis. The first factor investigated in this thesis relates to the impact of operational conditions (centrifugation intensity and time) on the recovery of HSV-DIS particles. The primary stage centrifugation step was studied using both HSV-DIS and wild type HSV particles with the aim to optimize the operational conditions in order to maximize viable titre of HSV-DIS. In the case of a swing-out centrifuge rotor 80% virus recovery was achieved after 90 min at 26000g. The 20% loss of virus was attributed to damage to the viral envelope by over-compaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed-angle centrifuge rotor operating at 26000g. Another major factor currently limiting the titre of disabled HSV (HSV-DIS) is that disablement reduces viral release into the culture medium to about 5% of the total viral particles present in the system. This is significantly less than the 50%, which are typically reported for wild type HSV. It has been shown that a large fraction of disabled enveloped HSV particles remain attached and/or closely associated to the surface of the cells. Different physical and chemical methods have been reported in the literature to improve the release of HSV-DIS particles into the medium but much remains to be elucidated. The second part of this thesis proposes a novel physical method and assesses its use to enhance the release of HSV-DIS particles. The method depends on the application of controlled shear to re-suspended pellets of cellular material carrying viable HSV-DIS particles. The shear experiments were carried out in the gap between a pair of co-axial cylinders, designed and constructed as part of this thesis. Pelleted infected cells recovered from a primary centrifuge stage was subjected to a range of shear rates from 3.69 x103 s-1 to 51.3 x103 s-1. Plaque assays and Polyacralamide Gel (PAA) analysis indicated that the application of a high shear rate, typically of the order of 40x103 s-1, for a period of approximately one minute increased release of HSV-DIS particles. These conditions increased the viable titre in the supernatant up to 30x (statistically standardized data), compared to control (no shear) experiments. FACS analysis carried out on sheared and un-sheared samples confirmed that in addition to viable viral particles, the supernatant contained cellular debris. Comparison of FACS data between sheared samples recovered from the supernatant with data obtained from the supernatant of the original culture (primary recovery stage), indicated that the supernatant material obtained after shearing of the resuspended material had the potential to be further processed into ultra-pure disabled HSV for use in clinical trials. The combination of controlled shear for release of HSV-DIS particles with centrifugation carried out under optimum conditions has the advantage of simplicity and scalability. A particular advantage of the shear release of viral particles proposed in this study is that no chemical agents are used in the release process. This will also make downstream purification and process validation easier.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Impact of Engineering Flow on the Release and Recovery of Disabled HSV from BHK Cells
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Applied sciences; Herpes virus
URI: https://discovery.ucl.ac.uk/id/eprint/10099627
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