Kee, G.K.; (2009) A rational approach to the development of future generation processes for lipoprotein VLP vaccine candidates. Doctoral thesis , UCL (University College London).

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Abstract

Lipoprotein VLPs, known also as lipid-envelope VLPs, are expressed intracellularly in yeast, localised on the host endoplasmic reticulum (ER). The main challenge in production lies in the complexity of the recovery and purification process. Using the Hepatitis B surface antigen (HBsAg) as the VLP model, strategies for exploiting the full potential of primary recovery operations to raise the overall process efficiency and throughput were investigated. Detergent-mediated liberation of HBsAg from the host endoplasmic reticulum is a critical step which influences product quality and yield and defines the characteristics of the resulting process stream. Screening studies established that Triton X-100 gave superior performance if concentrations were maintained within the range of 0.2% v/v to 0.5% v/v. Concentrations above this threshold led to HBsAg delipidation and loss of antigenicity. Increased levels of co-liberated host protein and of lipid contamination which resulted in poorer ultrafiltration performance were also observed. PEG and ammonium sulphate precipitations were investigated to reduce the level of host protein and lipid contaminants. In each case, although significant product enrichment was achieved, product loss of up to 50% was incurred owing to the difficulty of PEG precipitate recovery and resolubilisation and the lack of selectivity of the ammonium sulphate agent. A novel selective product recovery methodology was developed in which an additional centrifugation step was introduced post-cell disruption but prior to the addition of detergent. HBsAg associated with the ER was pelleted allowing bulk cytosolic contaminants in the supernatant to be eliminated. This approach was further enhanced by the use of moderate homogenisation pressures in the region of 400 bar. Recovery of active HBsAg improved by ~20% under lower shear conditions and a further ~40% reduction in the level of host lipid contaminants was observed. The benefits of the proposed process on a downstream chromatography step include better product capture and improved step yield. The scalability of the selective recovery method relies on the dewatering and clarification performance. A CARR tubular type centrifuge was identified to be the most suitable equipment type for this purpose. Ultra scale-down centrifugation experiments were employed to predict suitable operating conditions for a pilot-scale process which was subsequently carried out and successfully verified that the selective recovery methodology remained feasible upon scale-up. Overall, this study has demonstrated how the adoption of a rational approach to the design of primary purification operations can lead to significant improvements of downstream purification performance.

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