Uncovering Growth‐Inhibitory Metabolic Byproducts in HEK293 Fed‐Batch Cultures and Strategies for Their Control

Human Embryonic Kidney 293 (HEK293) cells are currently one of the preferred host cell lines for the production of biologics, specifically, AAV-based viral vectors. These fast-growing cells consume significant amounts of nutrients and often convert them into byproducts such as lactate and ammonia. In fed-batch cultures, accumulation of lactate and ammonia to high levels can inhibit cell proliferation. In this study, we demonstrate that lactate and ammonia accumulation alone doesn't fully explain the growth inhibition observed in HEK293 fed-batch cultures. Growth inhibition was noted even when the residual levels of these byproducts were well controlled. Instead, we show that several previously unknown compounds accumulate in HEK293 cell fed-batch cultures, some of which can inhibit HEK293 cell growth either individually or synergistically. Many of these newly identified compounds are intermediates or byproducts of amino acid catabolism. When residual levels of the source amino acids for these novel byproducts were controlled in the low concentration range (∼1 mM) in HEK293 fed-batch cultures, lactate accumulated to higher levels, causing growth inhibition. This prompted the use of High-end pH Delivery of Glucose (HIPDOG), a control strategy that limits lactate production by keeping low residual concentrations of glucose. In HIPDOG cultures, controlling the source amino acids at low concentrations resulted in lower accumulations of the corresponding growth-inhibitory byproducts when compared to the control HIPDOG conditions with typical levels of amino acids. This led to higher viable cell densities (VCD) and viabilities in low amino acid conditions. Strategies that reduce byproduct accumulation, whether classical or novel byproducts, in HEK293 fed-batch processes can result in enhanced VCDs, potentially leading to higher volumetric productivities.