Maybury, John Paul; (1999) Scale-down of a bioprocess sequence for the recovery and purification of an intracellular protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The performance of three scale-down unit operations for the recovery of an intracellular protein is examined as a tool to help accelerate process synthesis and development. Operations studied include, high pressure homogenisation, fractional protein precipitation, and disc stack centrifugation. The target protein is alcohol dehydrogenase from bakers' yeast (Saccharomyces cerevisiae), a typical intracellular protein. The effectiveness of the scale-down process in predicting pilot scale performance is shown. It is compared with the alternative small scale system, laboratory scale. Unit operation performance is examined individually and also in a process context. High pressure homogenisation performance is scaled down by maintaining the valve geometry, the cell concentration and the pressure drop across the valve. The scale-down high pressure homogeniser accurately predicts both product release and the resultant cell debris particle size distributions of pilot scale for the range of conditions examined. Scale-down protein release values are within 6% of pilot scale and the maximum error in the prediction of the mean debris particle size is less than 8%. Disc stack centiifuge scale-down is achieved by modifying a pilot scale machine. Both the liquid and solid hold-up of the centrifuge bowl is reduced whilst still maintaining the ability to perform intermittent solids discharges. Scale-down is achieved in stages using a series of interlocking inserts to suit particular applications. Maximum scale-down gives a 76% reduction in the separation area and a bowl volume reduction of 70%. Separation performance of the scale-down machine closely follows the full scale version when using particulate streams of polyvinyl acetate and yeast cell debris. The particle size distribution of fine material in the supematant stream closely predicted the pilot scale. Improved recovery performance with scale-down is seen with protein precipitates although this is small when recovery efficiencies are high. Protein precipitations are scaled down by maintaining similar vessel geometiy and the mean velocity gradient, G, experienced in the vessel. With the ammonium sulphate precipitant, protein and ADH solubilities are independent of scale. With the polyethylene glycol precipitant there is over-precipitation with increasing scale. Ammonium sulphate precipitates formed at all scales (0.9-40 L working volume) give similar particle size distributions. Both ammonium sulphate and polyethylene glycol precipitates formed at scale-down (3.6 L working volume) give comparable clarification efficiencies to pilot scale. With the ADH process sequence, the scale-down process predicts an overall product yield of 19% which is a good indication of pilot scale performance (11%). The laboratory scale process greatly over-predicts the yield (72-76%). The main reasons for disparities are differences in centrifuge performance.

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