The upstream processing of E. coli has seen considerable improvements in product titres in recent years, however in contrast, downstream processing remains challenging and represents a significant bottleneck in the production of biopharmaceuticals. This research uses established ultra scale-down (USD) methods as a platform to investigate the feasibility of using polyethyleneimine (PEI) as a flocculant and precipitant in the primary recovery of antibody fragments (Fab') from E. coli fermentation. The overall aim was to maximise product titre without compromising product quality, process costs or time. Two alternative primary recovery approaches were compared to the standard Fab' primary recovery stages (centrifugation, periplasmic extraction, centrifugation and depth filtration), which were modified based on the extent of cell lysis in the fermentation broth. A range of analytical techniques were used to characterise the efficiency of the separation, including DNA concentration, total protein concentration and product concentration. Mass spectrometry experiments were also carried out to assess whether the use of PEI affects the conformation and hence activity of the antibody fragments. This paper shows that by using tangential flow filtration (TFF) in combination with PEI, it was possible to achieve a 40% greater Fab' titre by running the fermentation to maximum productivity, whilst reducing DNA and host cell protein in the supernatant by 30% through precipitation with PEI. The use of the TFF device also simplified the primary recovery steps by removing the need for three unit operations; centrifugation, periplasmic extraction and depth filtration, whilst obtaining equivalent or greater separation efficiency through flocculation of cell debris with PEI. This intensified primary recovery process would also considerably reduce capital and operating costs at large scale. The data presented demonstrates how a process integration approach can yield step-changes in the improvement of the traditional primary recovery process, and shows promising results on the novel use of flocculation in combination with tangential flow filtration as an alternative process design that can achieve greater product titres and simplify the primary recovery process.

Investigating the use of Polyethyleneimine (PEI) in combination with tangential flow filtration for the primary recovery of antibody fragments from E. coli fermentation using ultra scale-down techniques

253rd National Meeting (2017)

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