Murrell, Nicholas James; (1998) An engineering study of the recovery of shear sensitive biological materials by high speed disk stack centrifugation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Advances in genetic engineering have led to the ability to express a product protein to specific locations within an organism. Of particular interest is expression to the periplasmic space, the area between the outer membrane and the inner (cytoplasmic) membrane. The target protein is then correctly folded to it's active form. This avoids an inefficient re-folding stage in the processing chain and enables the use of selective disruption techniques such as lysozyme treatment to release only the contents of the periplasmic space, thereby simplifying later purification. Such genetic manipulation however results in a much weakened cell which is liable to disruption by conventional cell harvest techniques such as disk stack centrifugation. It is the processing of such materials that forms the focus of this thesis. The initial phase of the work focuses on establishing a periplasmic system with high levels of expression. The effect of yeast extract addition to a defined fermentation media in decreasing segregational plasmid instability and thus increasing product titres is investigated. The yeast extract is found to be preferentially metabolised and thus induce diauxic growth with the associated lag phase allowing an increase in plasmid copy number and hence a higher product titre (from 15 I.U. min-1 ml-1 on a defined media to 100 I.U. min-1 ml-1 using yeast extract). High speed disk stack centrifugation is the unit operation of choice for cell harvest because of its' continuous nature of operation. Such machines though are known to expose material to significant shear fields in the feed (acceleration) zone at the entrance to the bowl which results in disruption and, in the case of periplasmic systems, product loss. The theoretical flow regime within the feed zones of both a standard semi-hermetic and an experimental hydro-hermetic (soft shear) feed disk stack centrifuge are considered and the areas within the feeds causing disruption identified. Estimates are made of the shear rates developed in both machines. The degree of disruption to a periplasmic expressing organism is characterised for both designs. The standard bowl was found to disrupt 15% of cells with release of up to 30% of product at optimal flow rate for good recovery. Operation with a soft-shear design gave figures of 2% and 7% respectively. The effect of varying flow rate and rotational speed upon cell disruption is investigated, with an increase in disruption being observed with rotational speed and an increase with flow rate followed by a decrease as flooding occurred in the standard design. The results of this work were used to design lab scale mimics based on both a spinning disk and a power dissipation disruption mechanism occurring within the centrifuge feed zones. These were then tested with whole cells to examine their utility in predicting product loss during a cell harvest process and shown to reproduce the disruption seen within the centrifuge feed zones. To examine the generality of the scale down approach tins study was then extended to shear-sensitive polyethylene glycol protein precipitates produced from yeast homogenate. The results of this study will enable the prediction of the levels of disruption occurring to a biological material during passage through a disk stack centrifuge. A greater understanding of the mechanisms causing such disruption has been achieved which can be utilised to optimise operating conditions to minimise damage and hence maximize recovery.

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