Boychyn, Robert Michael; (2000) Scale-down principles for the accelerated design of protein purification processes. Doctoral thesis (Ph.D.), University College London.

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Abstract

With speed to market as a critical factor determining the economic success of a therapeutic product, bioprocess development must be carried out as efficiently as possible. This relies heavily on the ability to predict industrial-scale operation with only small (< 0.5 L) quantities of available feed; laboratory scale-down is superior to pilot- plant work in terms of speed, ease, and cost. Application of such a strategy is particularly relevant to early solid-liquid separation stages, where the performance of a centrifuge or filter may largely determine process yield and the quality of material to be delivered to subsequent guard filtration and chromatographic stages. Previous work focused on the modification of a disc stack centrifuge and laboratory prediction of clarification for shear-insensitive species such as cell debris. This thesis extends this work by developing laboratory-scale methods to mimic sediment dewatering and recovery of shear-sensitive protein precipitates in continuous-flow centrifuges. Initial experiments conducted with a multichamber-bowl, resulted in a great difference between predicted and pilot clarifications (50% lower) due to particle break-up occurring in the high-velocity entrance zone of the pilot centrifuge. The hydrodynamic forces in this region were analysed by computational fluid dynamics and reproduced in a small highspeed rotating disc device. Exposing suspension to the device prior to laboratory centrifugation permitted accurate prediction of pilot clarification. This technique was translated to other continuous centrifuges (disc stack, CARR, production multichamber-bowl). Importantly, the performance of the production centrifuge was more accurately predicted by the scale-down process than the pilot one. A simpler scaling tactic of constant tip velocity of the distributor in the centrifuge feed zone was also examined and found to be a good predictor of large-scale clarification. Conventional filtration (with precoat and body feed) was investigated as an alternative primary separation step. A production rotating vertical leaf filter was scaled down by transformation of a laboratory-batch, Nutsche pressure-filter; the continuous filter resulted in cakes of more uniform composition with lower specific resistances. Filtration gave significantly better clarification and sediment dryness than centrifugation. Finally, chromatographic performance was shown to depend moderately on the extent but principally on the type of solid-liquid separation, with centrifugation samples resulting in significantly greater column dynamic capacities than filtrates, which was not predicted by the response of a guard (cartridge) filter.

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