Characterising Chinese Hamster Ovary Cell Extracellular Vesicle Production in Biopharmaceutical Manufacturing

Extracellular Vesicles (EVs) are lipid bilayer bound particles containing several classes of protein, lipid and nucleic acid that are secreted by cells. Depending on the subclass of EV in question, they range from 30 – 1,000nm in size. Over the last decade, their utility in cell-to-cell communication has been described in detail in literature. This has ranged from use as biomarkers in cancer to tissue regeneration in stem cell therapies. Chinese Hamster Ovary (CHO) cells are one of the most commonly used host cell lines in biopharmaceutical manufacturing; particularly in the case of monoclonal antibody (mAb) production. They can be grown to large cell densities, are less susceptible to viruses which infect human cells and can glycosylate proteins in a human-like-manner. These characteristics, amongst many others, have enabled industrial processes to be created that maximise mAb production in CHO cells. Whilst it is known that CHO cells secrete EVs, little is understood about the influence of EVs in a biopharmaceutical manufacturing context or what factors impact EV secretion. This thesis established if CHO cells utilised EVs in biopharmaceutical manufacturing by sampling EVs from different time-points in a CHO fed-batch process. This determined if the quantity, size or composition of those EVs change as a fed-batch progresses through different stages of growth. It also looked to see what impact EVs have on their recipient cells; particularly through characterisation of the EV RNA content. By comparing the EV profile of a non-producing host cell line to a producer cell line, differences in the RNA and lipid composition of EVs between cell lines were investigated. There was also an analysis of what factors drive EV biogenesis in CHO. This assessed whether a particular stage in the cell line development process impacts EV secretion or if there are distinct cellular characteristics which correlate with EV secretion. This was done both by physical quantification of EVs and by transcriptomic analysis of genes involved in EV Biogenesis. Lastly, the thesis looked to determine if CHO EVs could be used for cellular engineering purposes through promotion of cell growth or by enhancing single cell cloning efficiency. Taken together, this thesis shows that EV secretion in CHO is largely a conserved process. It reveals that while the EV RNA content may serve to provide anti-apoptotic stimuli, the nucleic acid composition remains largely consistent throughout the manufacturing process. Interestingly, the quantities of EVs secreted and transcriptional activity of EV biogenesis genes does vary considerably between cell lines; with a significant negative correlation with mAb secretion. There is also variation in lipid composition depending on the cell line the EVs are derived from. Cellular engineering attempts which involved using purified EVs to promote growth in low density cell cultures and survival of single cell clones were unsuccessful. Again, this further alludes to EV biogenesis in CHO being evolutionarily conserved with limited utility for biopharmaceutical manufacturing purposes.