Fischer, Emma Jane; (1996) Impacts of separation processes on protein recovery from cells and cell spheroplasts. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis uses studies on the recovery of a recombinant periplasmic α-amylase from Escherichia coli to illustrate process development for the recovery of biological products for purification. Fermentations have been carried out in batch mode using defined or complex medium at up to 450 L scale, and also at 20 L scale in fed-batch mode using a defined medium. Centrifugal whole cell recovery led to cell damage in each of the types of centrifuges tested. Damage was highest in the tubular bowl centrifuge, intermediate in a disk stack centrifuge, and least in a multichamber centrifuge. The breakage in the disk stack centrifuge occurred mainly in the solids discharge. There was little breakage during microfiltration of a complex medium broth. 0.2 µm or 0.8 µm ceramic membranes were used to filter the spheroplast stream produced by a combined lysozyme-osmotic shock treatment on the recovered cells. No additional release of retentate protein was seen during microfiltration. Transmembrane pressure appeared to have little impact on the flux, but higher pressures reduced the transmission levels. The α-amylase transmission was higher than total protein transmission with the 0.2 µm membrane. No α-amylase selectivity was seen with the 0.8 µm membrane. The transmission performance during the trials has been correlated with the flux by means of a 'transmittance' term. Permeate backpulsing has led to a significant improvement in the performance of a 0.2 µm filtration. An investigation of the properties of the pulse has shown that there exists an optimum pulse frequency, and that a reverse flow of liquid is required for improved performance. Scale-up of microfiltration using constant wall shear rate has been demonstrated. Centrifugal removal of cell spheroplasts showed a lower degree of protein release than with the whole cells. However chromatography grade material could only be produced by centrifugation at low flowrates.

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