Liu, Shuchang; (2024) Assessment of CV-1 as a Production Cell Line for Large Scale Manufacturing of a Lister Strain Oncolytic Vaccinia Virus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Oncolytic Vaccinia virus (OVV) has emerged as a promising therapeutic agent for cancer treatment. However, the development of an effective manufacturing process for OVV has yet to be fulfilled. This thesis explored and evaluated the potential of using CV-1, a continuous cell line derived from monkey kidney with an indefinite lifespan, to develop a scalable production process for an oncolytic vaccinia virus. CV-1 cells have been widely used for vaccinia virus production in research labs. The cell line was originally maintained in serum-containing medium (SCM) statically. In this study, the cell line was adapted to grow in two serum-free media, VP-SFM and OptiPRO, with OptiPRO proving to be the most suitable. The adapted cell line, CV-1- OPT, exhibited the shortest doubling time, highest plating efficiency, a recovery ratio comparable to that of SCM, and a saturation density 85% of that observed in SCM. CV-1-OPT also demonstrated effective proliferation on microcarriers, with cell densities reaching similar levels in batch cultures regardless of initial seeding densities, and elevating up to 1.87×10⁶ cells/ml when intermittent feeding strategies were employed. The production of an oncolytic vaccinia virus, VVL-15 RFP, was studied using the previously developed microcarrier-based CV-1-OPT suspension culture system. The cells were infected during their mid-late to late exponential phase when the density was between 9.27×10⁵ to 1.52×10⁶ cells/mL, with a multiplicity of infection (MOI) ranging from 0.1 to 1. The maximal specific viral productivity achieved was 71.9 pfu/cell. Infection at a low cell density with a high MOI led to a reduction in production time. The study results demonstrate the successful adaptation of CV-1 cells to OptiPRO SFM, their efficient proliferation on Cytodex-1 microcarrier and capability in supporting VVL-15 RFP expansion. These promising findings suggest that CV-1 cells could potentially serve as a feasible option for large-scale production of oncolytic VV.

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