Cell line development for continuous high cell density biomanufacturing: Exploiting hypoxia for improved productivity

Abstract

Oxygen deficiency (hypoxia) induces adverse effects during biotherapeutic protein production leading to reduced productivity and cell growth. Hypoxic conditions occur during classical batch fermentations using high cell densities or perfusion processes. Here we present an effort to create novel engineered Chinese hamster ovary (CHO) cell lines by exploiting encountered hypoxic bioprocess conditions to reinforce cellular production capacities. After verifying the conservation of the hypoxia-responsive pathway in CHO cell lines by analyzing oxygen sensing proteins HIF1a, HIF1β and VDL, hypoxia-response-elements (HREs) were functionally analyzed and used to create hypoxia-responsive expression vectors. Subsequently engineered hypoxia sensitive CHO cell lines significantly induced protein expression (SEAP) during adverse oxygen limitation encountered during batch fermentations as well as high cell density perfusion processes (2.7 fold). We also exploited this novel cell system to establish a highly effective oxygen shift as innovative bioprocessing strategy using hypoxia induction to improve production titers. Thus, substantial improvements can be made to optimize CHO cell productivity for novel bioprocessing challenges as oxygen limitation, providing an avenue to establish better cell systems by exploiting adverse process conditions for optimized biotherapeutic production.