A study of the use of combined precipitation and selective flocculation for improving the primary recovery of virus-like particles.

摘要

Virus-like particles (VLPs) are non-infectious protein aggregates produced intracellularly by the yeast Saccharomyces cerevisiae. Recombinant DNA techniques have been used to produce VLPs with a chosen foreign protein expressed on the particle surface. These hybrid particles have potential as vaccines, diagnostics and therapeutics. In the industrial manufacture of VLPs, high pressure disruption is used to release the product. Subsequent clarification of the homogenate by disk stack centrifugation and depth filtration prior to column chromatography results in losses of 60-70 %. The objective of this study was to improve VLP recovery over these primary stages by the use of selective steps to remove key contaminants such as nucleic acids and total protein, while retaining the basic solid-liquid separation equipment. Laboratory experiments were performed using baker's and VLP- expressing yeast homogenate to assess the use of borax flocculation followed by centrifugation to remove cell debris. Results confirmed the ability of borax to improve centrifugal clarification of crude homogenate by selective flocculation of ceil debris particles. Borax had no effect on VLP and soluble protein levels. The resulting supernatant was treated with PEG of varying molecular weights to precipitate the VLPs present. The degree of precipitation increased with PEG concentration and degree of polymerisation, and in more acidic conditions. Precipitation was insensitive to temperature in the range 5-30°C. Fractionation diagrams were used to determine the selectivity of the precipitation; VLPs were found to be brought out of solution in preference to soluble protein and nucleic acids, conferring a degree of selectivity. VLP-containing yeast was produced by 1500 litre fed-batch fermentation, producing biomass levels comparable to industrial fermentations. Pilot- scale trials of combined borax flocculation and PEG precipitation were carried out using this material, with solids recovery by disc stack centrifugation. The new process route gave an approximate three-fold reduction in process losses whilst remaining compatible with the existing process equipment.