Martinez, Michael; (2021) Scale-up of continuous monoclonal antibody precipitation. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

The scale-up of protein precipitation processes proves to be a challenging task due to the complexity of the reactions and transport processes involved. A good understanding of the molecular processes underpinning precipitate formation and the reaction kinetics are therefore required in order to devise a scale-up strategy. The doctoral project was first set out to establish micro-mixing as an engineering tool for the scale-up of antibody precipitation from cell culture, and secondly to design a downstream process with the goal of purifying a therapeutic mAb to clinical grade levels. Studies were first conducted in batch and transferred to a continuous process, with the scale-up approach focusing on the latter. Interactions between precipitation conditions and centrifugal recovery were then examined by employing an ultra scale-down (USD) methodology to mimic large-scale centrifugation. The downstream process design was on the basis of integrating precipitation with non-affinity chromatography steps to avoid the cost of affinity chromatography. Precipitate formation in batch and continuous settings was governed by the mixing at the molecular scale, which determined the final particle properties. Based on this, the mean energy dissipation rate for a continuous precipitation process proved an effective scale-up criterion, enabling high process throughputs relative to batch operation. The strength of protein precipitates, as evaluated by exposing particles to turbulent shear in a rotating disc device, was shown to correlate with particle fractal dimensions. Despite excellent precipitate solids removal from the USD methodology, these could not be predicted by disc-stack centrifugation. Differences in hindered settling between the systems were proposed to explain this observation which suggests routes to resolve this scale-up challenge. To provide an integrated DSP solution for therapeutic mAbs processes anion exchange and mixed-mode chromatography steps subsequent to precipitation were designed. Parameter ranges were studied to identify the optimal conditions in maximising antibody yield and HCP removal. Using optimal conditions, precipitation and anion exchange demonstrated an 18-fold removal in HCPs, whilst precipitation and mixed-mode provided a 40-fold removal. For a three step process comprising the sequence precipitation, anion exchange and mixed mode, an overall HCP removal of 260-fold was seen; however such levels remain at least 38-fold higher than the typical specification of a clinical grade product. This therefore necessitates further optimisation in one or more steps.

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