Modulating epigenetic and post-transcriptional regulation for increased recombinant protein production in mammalian cells.

Recombinant protein based therapeutics, such as monoclonal antibodies have shown remarkable pharmaceutical market penetration over the past 10 years. There is also a large number of new biologics in development as well as a steady flow of blockbuster follow-on biologics expected to enter the market in coming years. However costs of biologics remain unsustainably high, often exceeding US$100K per person per year, putting considerable strain on healthcare providers and limiting use in 2nd and 3rd world countries. There is now a substantial need to reduce the production costs and increase product consistency. With bioprocess-based improvements reaching a plateau, process bottlenecks will require novel scientific solutions. The equation is simple, more high-quality protein is required to be produced in a shorter period of time, i.e. “more for less”. This thesis looks to investigate the application of various techniques that can assist in meeting the more for less challenge. This investigation covers the use of site-specific integration as an alternative approach to gene integration with the incorporation of vector engineering components such as chromatin remodelling elements and untranslated regions. While these vector augmenting elements show great promise in recombinant protein expression, further understanding into the applications of these elements are necessary to extract the maximum potential from mammalian expression systems. The thesis also investigates whether promoter methylation is affecting expression stability of a CHO (Chinese hamster ovary) cell expression system. This project aims to further understand these industrial relevant tools for the improved production of recombinant proteins. In this work we demonstrated that the inclusion of chromatin remodelling elements in combination with automated clone picking has the ability to significantly reduce cell line development time lines from 6 months to 6 weeks as well as providing significant improvements in expression levels. Top clones identified in this process were capable of producing over 200 mg/L in a simple batch study over 6 days. Analysis of both the 5’ and 3’ UTR (untranslated region) through methylation analysis and vector engineering has illustrated a combination of scientific and engineering approaches to further understand the impact of both regions on recombinant protein production. The screening of the hCMV (human cytomegalovirus) promoter CpG sites shows potential methylation sites prone to methylation, while vector based engineering was able to further current understandings in the use of UTR control regions for recombinant protein expression through the comparison of both mRNA and protein levels. The work illustrated in this thesis attempts to address some of the bottlenecks in current industrial processes. The characterisation of upstream vector design has real implications in a large-scale process; therefore we have successfully incorporated a number of key elements which have influenced final production yields. With the recent publication of the CHO genome, the growth of the industry will depend on applications of the new technologies and the ability to augment these highly complex mammalian systems