title: Investigation of Translational Reprogramming during Transient and Stable Expression of Monoclonal Antibodies in Chinese Hamster Ovary Cells
Creators: name: Godfrey, Charlotte
name: MedImmune
Publisher: name: University of Kent
Year: 2017

description: Translational reprogramming and mRNA translation e ciency greatly in uence global protein synthesis, cell proliferation and growth; important parameters in de ning recombinant protein expression yields. Polysome pro ling is a widely-used technique to analyse mRNA transla- tion and its e ciency that provides a snapshot of ribosomes loaded on mRNA transcripts at any particular time. A higher number of polysomes present on a given mRNA suggests that the mRNA is being more heavily translated than those mRNAs with few ribosomes. Fur- ther, a large pool of sub-polysomes (40S, 60S and 80S) compared to polysomes in a sample suggests low translational activity. Here, polysome pro ling has been applied to investigate translational reprogramming in multiple recombinant monoclonal antibody (mAb)-producing Chinese hamster ovary (CHO) cell lines, and to determine how reprogramming re ects the ability of such cells to proliferate and make recombinant proteins in stable and transient mAb expression systems, in batch and fed-batch culture mode. The impact of culture temperature on the polysome pro le and hence on reprogramming was also investigated in transient studies. Polysome pro ling revealed reprogramming di ered between recombinant cell lines. Those with the highest global translational e ciency generally had the fastest cell speci c growth rates, although total ribosome capacity did not directly relate to those with the fastest growth rates or mAb productivities. This suggests it is the ability to utilise available machinery that determines protein synthetic capacity. Recombinant cell lines with higher cell speci c produc- tivities generally maintained a higher polysome to monosome (P:M) ratio during stationary phase and had elevated recombinant mRNA copy numbers localised to translationally active heavy polysomes. In transient systems, the P:M ratio was maintained longer at reduced tem- perature cultivation and related to higher mAb yields being obtained. A number of endogenous transcripts were found to be more or less abundant on polysomes at di erent times of culture, indicative of changes in the cellular requirements of the encoded proteins. Such transcripts could be potential cell engineering targets to help tune the needs of the cell to the demands of a culture process or recombinant protein, or alternatively their untranslated regions harnessed to help preferentially load target mRNAs onto ribosomes. When upstream open reading frames (uORFs) or alternative translation start sites were engineered into recombinant transcripts a range of mAb expressions were observed allowing the tuning of mAb expression, including improvement over a standard untranslated region used industrially as a control. The ndings described in this thesis therefore reveal insights into the mechanisms involved in translational regulation and reprogramming in CHO cells during bioprocessing. These can be utilised for further improvement via targeted cell engineering strategies, cell line screen- ing approaches or modi cation of recombinant transcripts for enhanced industrial host and recombinant cell lines.