Schofield, DM; (2016) Synthetic biology approaches to bioprocess intensification in bacterial and mammalian production platforms. Doctoral thesis , UCL (University College London).

Abstract

The controlled production of recombinant proteins and their high purity recovery are key goals of biochemical engineering, and can be met by concerted design of cellular hosts using synthetic biology tools. This thesis addresses five process areas with biological solutions.DNA is a major impurity in mammalian process streams, and can be removed by adding nuclease to the feedstream. Here, a HeLa cell line which encodes a recombinant nuclease is successfully adapted to serum-free growth and its activity is characterised. Optimising protein induction within E. coli is a time consuming process; here a synthetic gene network which is predicted to continually produce a recombinant protein once activated is assessed and the host cell line, culture environment, and analytical tools developed.Previously work with a biotherapeutic protein had failed to produce a dimer in a high yield or pure form from E. coli inclusion bodies. A cysteine residue was substituted into the sequence for dimerisation, and a bench scale production process yielding 108 mg of 95% pure dimer was developed. The monomeric form was chemically crosslinked to produce an enhanced stability dimer. DNA increases homogenate viscosity; here, an E. coli strain expressing a SRP-trafficked nuclease is constructed and transformed with a plasmid encoding an SEC-trafficked antibody fragment (Fab’); this strain autohydrolyses DNA, but maintained a high Fab’ yield. This contrasts with a previously constructed SEC-trafficked nuclease, which reduced the yield and increased cell lysis. Proteomic analysis showed that utilising two translocation pathways depleted periplasmic proteins and a deterministic model suggested a maximum of 6% of the total periplasm volume could be occupied by recombinant proteins, above which lysis occurred.In order to test this threshold, a promoter-engineered variant of the Fab’ production strain was constructed. Bench-scale fermentations showed improved product retention, cell viability, and validated the 6% hypothesis.

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