Understanding Chinese hamster ovary cell translation at sub-codon resolution

Chinese hamster ovary (CHO) cells are the dominant mammalian expression host for recombinant therapeutic protein production. In terms of manufacturing efficiency, much has been accomplished in areas such as optimised transgene design and cell line development. Since the publication of the Chinese hamster genome the field has gained a more refined understanding of the relationship between the CHO biological system and desirable bioprocess traits. Despite the central importance of protein synthesis, few studies to date have focussed on characterising translation in CHO cells. The goal of this thesis is to evaluate the utility of ribosome footprint profiling (Ribo-seq) to further improve our understanding of CHO cell biology and highlight routes towards enhanced biopharmaceutical manufacturing. A key aspect of this work is the combination of multiple translation inhibitors for Ribo-seq to enable the simultaneous analysis of translation initiation and elongation for the first time. The availability of these data enabled the identification of previously uncharacterised open reading frames (ORFs) including those non-AUG start codons. Novel ORFs comprised of N-terminal extensions of canonical proteins, ORFs found in genes previously thought to be non-coding and those found in the 5’ leader sequence of mRNAs (i.e. upstream ORFs). Through the use of Ribo-seq and RNA-seq data, these upstream ORFs were found to have a repressive effect on the translation efficiency of the main ORF. In addition, following comparison of CHO cells at day 4 and day 7 of cell culture as well upon a reduction of cell culture temperature, genes undergoing differential translation were identified. A number of these genes did not have a corresponding change in gene expression, confirming that Ribo-seq can provide an additional dimension compared to using RNA-seq in isolation. Ribosome profiling has further enabled the computation of transcriptome wide decoding times for each codon, and revealed influence of codon context on translational rate. These data provide a potential route towards more efficient codon optimised transgene sequences. Perhaps the most striking finding of this work is the identification of thousands of novel small open reading frames (sORFs) predicted to encode microproteins (i.e. proteins < 100aa). Host cell protein analysis, revealed that 8 microproteins were present in adalimumab, confirming that microproteins are a novel class of potential process related impurity. In summary, ribosome footprint profiling is a powerful analytical method for improving the annotation of the CHO cell genome, understanding CHO cell biology and identification of routes to improve not only the upstream process but also enhance the characterisation of the final drug product.