Seymour, Maile Elizabeth Kahawaluiaakalani; (2002) Process considerations in the extraction and recovery of plant virus vaccines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The plant virus cowpea mosaic virus (CPMV) has recently been developed as an antigen presentation system intended for future use in human vaccines. In this work, scale-down process studies of initial extraction and recovery of CPMV were carried out using wild type CPMV as a model for more valuable recombinant variants. Release of contaminating soluble protein was also measured. Process conditions used throughout were suitable for large scale production. Actual processing volumes were of the order of millilitres. Fresh and frozen CPMV-infected leaf material was disrupted by homogenisation in batch and continuous modes and by bead milling. Rheological measurements of the homogenate indicated shear thinning behaviour with constant minimum viscosity of 0.03 Pa s at typical process shear rates (>200 s-1). Particle size distributions (mode, 7 microns) showed that cells in the tough plant tissue had been efficiently disrupted. Yields from fresh and freshly frozen leaves were comparable, and extended deep-frozen storage of leaves for up to three years appeared to increase yields, to a maximum of 10.4 mg CPMV per g frozen leaf. Yields varied markedly between plant growth batches. Salt precipitation with ammonium sulphate (up to 3.1M) and neutral polymer precipitation with polyethylene glycol (PEG) (up to 25%), in single and double cut strategies, were used to recover CPMV from the leaf homogenate with and without prior clarification by low speed centrifugation. For ammonium sulphate, double cut precipitation from untreated homogenate gave the best yields and purification. For PEG, the best strategy was single cut precipitation from clarified homogenate. Both alternatives achieved a volume reduction of 20- to 25-fold from the homogenate to the precipitate pellet, 10-fold from leaf to pellet. The optimal scale-down process conditions determined for the wild type CPMV were applied to purification of a recombinant muc14-CPMV. Yields for the muc14-CPMV agreed well with those obtained for the wild type.

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