Perry, Christopher; (2021) Lentiviral vector stability and process purification based on modifications to the viral envelope. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Lentiviral vectors (LV) are potent tools in delivering genetic information for vac-cines and therapeutic benefits. Bioprocessing of LV remains challenging, with vec-tor generation dependent on transient transfection methods demanding high costs which are subject to batch variability. Stable producer cell lines can be employed as alternatives to promote consistent vector generation; however, the LV envelope protein must be carefully selected, with the common envelope, VSV-G, unsuitable due to cytotoxicity. Alternative envelope proteins such as RDPro and Cocal-G have been developed and their impact in bioprocessing evaluated herein. Various unit operations were studied to make comparisons of how envelope proteins affect the bioprocessing of LV. Herein, the development of a stable pro-ducer cell line with Cocal-G envelopes are described and evaluated against existing RDPro cell lines. Following this, the performance of LVs with differing envelope proteins in filtration and chromatography was investigated. The generation of stable producer cell lines with Cocal-G envelopes indicated superinfection related instability, which was ameliorated with small drug treatment, permitting adherent culture scale up over five days, generating a total of 1.92X109 TU total. During filtration, envelope and filter chemistry was of main effect with polyvinylidene difluoride membranes and VSV-G envelopes were shown to be most effective for vector transmission irrespective of filters and envelopes. During shear characterisation, all vectors were resistant up to 106s−1 shear rate, typical of most process equipment, in the ultra-scale down shear device even in the presence of air-liquid interfaces. In the membrane shear device, losses, likely from membrane adsorption, are evident with RDPro least affected. Generally, increased disc speed improved recovery although the ratio of transducing units to vector particles de-creased. A design of experiment method for anion exchange revealed weaker inter¬action of Cocal-G enveloped LVs in QA and DEAE ligand monoliths that allowed lower salt concentration to elute (1.1 M compared to 1.3 M). Affinity chromato¬graphic purification of VSV-G vectors by small protein ligands, based on the LDL-R binding domain, captured and depleted vectors in suspension, however elution was not successful with pH decrease although incubation with bead fractions achieved approximately 55% recovery. Results demonstrated different performance of vectors across unit operations which are influenced by envelope choice. The findings reported herein should assist in the achievement of scalable and robust processes for the utilisation of lentiviral vectors.

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