Enhancing secretory pathway function to improve production of recombinant therapeutic proteins

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Protein-based therapeutics are a strategically-important and rapidly expanding component of the industrial biotechnology sector, and are typically manufactured via bioprocessing using mammalian host cells engineered to produce high levels of the therapeutic protein (Dumont et al., 2016). A critical step in the production of a novel biopharmaceuticals is the generation of a clonal cell line that stably expresses and secretes the protein in large amounts. InstiGRO, a media supplement developed by SAL Scientific, greatly improves the efficiency of limiting dilution cloning of DHFR- CHO cells by improving cell survival and subsequent proliferation. Preliminary results from the co-supervisor's laboratory in collaboration with SAL Scientific led to the hypothesis that InstiGRO is particularly beneficial for generating lines producing 'difficult-to-express' proteins. In addition, lines selected using InstiGRO appeared to produce more protein. Given that secretory pathway capacity can be a limiting factor in biopharmaceutical protein production, this raises the possibility that InstiGRO promotes secretory pathway function in CHO cells so that their ability to handle and traffic 'difficult' proteins is enhanced. This project will investigate how 'easy' and 'difficult' cargo proteins are trafficked through the secretory pathway in a range of cell types, including CHO lines that are the 'workhorse' cell line for protein production by the biopharmaceutical industry (Dumont et al., 2016). We will use a novel live cell imaging approach (Boncompain et al., 2012) combined with biochemical analysis to follow the kinetics and trafficking of each cargo from the endoplasmic reticulum (ER), via the Golgi apparatus, to the plasma membrane (PM). We will determine which type of membrane carriers are used for Golgi-to-PM transport, and which microtubule motor of the kinesin superfamily drives their movement (Ruane et al. 2016), as both can differ between cargo proteins. Comparing CHO lines with other cell types commonly used for trafficking studies (HeLa, Vero, COS) across a range of cargoes will reveal if there are adaptations of the secretory pathway that makes CHOs particularly well-suited to protein production, and pinpoint bottlenecks in the trafficking of difficult cargoes that could be targets for future optimisation. With this characterisation complete, we will then determine how InstiGRO affects the secretory pathway and protein production levels, and whether these effects are enhanced to a greater extent for 'difficult' vs. 'easy' proteins.