A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) routinely generate lines. Here we describe development of a system based on metabolic requirement CHO proline, and that uses pyrroline-5-carboxylase (P5CS) complement this auxotrophy. Firstly, showed can be used have growth kinetics in proline-free medium similar those parent line, CHOK1SV GS-KO™ grown proline-containing medium. As previously described how engineering lipid metabolism harnessed enhance protein productivity cells, then P5CS re-engineer by over-expression either sterol regulatory element binding 1 (SREBF1) stearoyl CoA desaturase (SCD1). The with re-engineered proline consistent P5CS, SCD1 SREBF1 expression across 100 generations. Finally, show GS systems together. A vector containing light heavy chains for mAb was super-transfected into over-expressing from vector. resulting stable transfectant pools achieved higher concentration at harvest model difficult express than host. This demonstrates concomitantly enable line genetic expression.