Manipulation and engineering of lipid metabolic pathways in CHO cells to enhance processability of cell culture supernantants

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The CHO cell is the current industrial system of choice for the expression of complex, post-translationally modified recombinant biopharmaceutical proteins Current CHO host cells have been developed for the commercial manufacture of monoclonal antibodies in particular, however many new protein biologics are difficult to express and/or cellular components can present problematic downstream processing issues. One area which has received little attention in CHO cell biology is lipid biosynthesis and the impact of lipids on cell phenotype (cell growth, product yields). Lipids are the major component of cellular membranes, are integral to energy metabolism, cell signalling, cell growth and survival, organelle formation, and transport/secretion via vesicle formation and trafficking. At Kent we have shown that manipulation of lipid biosynthesis can result in increased secreted recombinant protein production and changes in the morphology and nature of vesicles released from CHO cells. The proposed project will elucidate key genes and pathways in lipid metabolism in CHO DG44 and CHOK1 host cell lines and then manipulate these, determining the subsequent impact on culture viability, cell growth, yield and quality of secreted biotherapeutic protein, vesicle (specifically exosome) production and on primary downstream processing events. Specifically the programme of research will involve; Work package (WP) 1: Perform siRNA and CRISPR knockdown/out experiments of key lipid metabolism targets in the CHO host and recombinant biotherapeutic producing cell lines and confirm knockdown/out by qRT-PCR, genome sequencing and western blotting. WP2: Assess the impact of knockdown/out of targets confirmed in WP1 on cell phenotype (growth, recombinant protein yield and quality), lipid metabolism, vesicle/exosome production and primary recovery steps from cell culture supernatants. WP3: Generate novel, engineered CHO host cell lines with target genes either knocked down or over expressed based on WP2 results (select top 2-5 targets) and assess these for recombinant protein production, exosome production and primary recovery.